Chronic treatment regimen using glucagon-like insulinotropic peptides

ABSTRACT

The present invention encompasses a method of treating a disease by maintaining chronic steady state serum levels of a GLP-1 compound within a specified range.

[0001] This application claims the benefit of U.S. ProvisionalApplication No. 60/255,251, filed Dec. 13, 2000, No. 60/295,655, filedJun. 4, 2001 and No. 60/298,652 filed Jun. 15, 2001.

1. FIELD OF THE INVENTION

[0002] The present invention relates to a chronic treatment regimenusing glucagon-like insulinotropic peptides in pharmaceutical articlesof manufacture and methods.

2. BACKGROUND INFORMATION

[0003] The intestinal hormone glucagon-like peptide-1 (GLP-1) showsgreat promise as a treatment for type 2 diabetes due to its ability tostimulate insulin secretion, lower glucagon secretion, inhibit gastricemptying, enhance glucose utilization, and induce appetite suppressionand weight loss. Further, pre-clinical studies suggest that GLP-1 mayalso act to prevent the β-cell deterioration that occurs as the diseaseprogresses. Perhaps the most salient characteristic of GLP-1 is itsability to stimulate insulin secretion without the associated risk ofhypoglycemia that is often seen when using insulin therapy and sometypes of oral therapies. When blood glucose levels drop to a certainthreshold level, GLP-1 is not active.

[0004] However, the usefulness of therapy involving GLP-1 peptides hasbeen limited by the fact that GLP-1(1-37) is poorly active, and the twonaturally occurring truncated peptides, GLP-1(7-37)OH andGLP-1(7-36)NH₂, have extremely short half-lives and are rapidly clearedfrom the circulation. Thus, research related to GLP-1 has focused on thedevelopment of GLP-1 analogs, GLP-1 derivatives, and formulationsthereof which provide a more extended time action. Despite much progressin this area of development, there are no published papers reporting onclinical data in humans using long-acting GLP-1 analogs or derivatives.Until the present invention, it has been unclear whether steady statelevels of a GLP-1 compound with a particular potency can be safelymaintained for a lengthy course of treatment and continue to provide thebenefits associated with the activities that have been elucidated forendogenous GLP-1.

[0005] Some short-term clinical studies with native GLP-1 which requirecontinuous infusion or frequent dosing suggest that high concentrationsof GLP-1 cause frequent nausea and vomiting. This has raised concernamong clinicians that these undesired effects will limit the dosage andthus, limit the efficacy even though the drug inherently may be capableof producing a greater effect.

[0006] There are several published clinical studies involvingadministration of native GLP-1(7-37)OH to patients by i.v. orsubcutaneous continuous infusion. See Naslund, et al. (1999) Am. J.Phys. 277(3):1-14; Deacon, et al. (1995) Diabetes 44:1126-1131;Toft-Nielsen, et al. (1999) Diabetes Care 22(7):1137-1143. The publishedstudies consistently use a dose between about 0.75 pmol/kg/min and 2.4pmol/kg/min for short periods of time. However, because GLP-1(7-37)OH israpidly degraded upon exposure to plasma, it is not always clear whatlevels of intact/active GLP-1 peptide are present in the plasma at agiven time point.

[0007] Naslund, et al., nevertheless, were able to predict plasma levelsof intact GLP-1(7-37)OH after continuous administration at a rate of0.75 pmol/kg/min for 180 min. The authors used a sandwichradioimmunoassy to detect both N-terminally degraded and intactGLP-1(7-37)OH. Plasma levels of intact GLP-1 were approximately 20picomolar for the last 120 min. of treatment. Using a similar assay,Toft-Nielsen et al. reported intact GLP-1 levels of approximately 10.2picomolar and 22.5 picomolar after administration of 1.2 pmol/kg/min and2.4 pmol/kg/min, respectively.

[0008] These studies, however, have not answered the question whetherplasma levels of intact and active GLP-1 can be achieved to achievetherapeutic benefit while at the same time avoiding or minimizing sideeffects such as nausea and vomiting. Similarly, these studies do notindicate whether plasma levels of active GLP-1 should be relatively flatduring treatment or whether peaks and valleys, which would mimic thephysiological state, would be preferred. Thus, despite considerableprogress, there remains a need to discover and understand what treatmentregimen leads to effective long-term therapy and whether such treatmentcan be maintained with minimal side effects such as nausea and vomiting.

[0009] Applicants have discovered that maintaining continuous plasmalevels of a GLP-1 compound in a specific range provides effectivetreatment. It is believed that the absence of peaks and valleys avoidsor minimizes side effects such as nausea and vomiting. Accordingly, thepresent invention provides a chronic treatment regimen which comprisesmaintaining continuous plasma levels of a GLP-1 compound within acertain range that avoids or minimizes side effects such as nausea andvomiting. The plasma levels encompassed by the present invention provideoptimal blood glucose control. Furthermore, this treatment regimenprovides long-term positive health effects including the inducement ofweight loss, improvement of β cell function, activation of dormant βcells, differentiation of cells into β cells, β cell proliferation, andthe maintenance of organ function.

[0010] The present invention encompasses a method of normalizing bloodglucose levels, preventing β cell deterioration, inducing weight loss,or treating a condition selected from the group consisting of:hyperglycemia, type 2 diabetes, obesity, stroke, myocardial infarction,catabolic changes that occur after surgery, and irritable bowelsyndrome, which comprises maintaining chronic steady state plasma levelsbetween about 60 picomoles/liter and about 200 picomoles/liter of aGLP-1 analog or derivative in a biologically active form having an invitro potency within two-fold the in vitro potency of Val⁸-GLP-1(7-37)OHwherein the GLP-1 analog or derivative is administered by subcutaneousinjection no more than once or twice every 24 hours.

[0011] The present invention also encompasses a method of normalizingblood glucose levels, preventing β cell deterioration, inducing weightloss, or treating a condition selected from the group consisting of:hyperglycemia, type 2 diabetes, obesity, stroke, myocardial infarction,catabolic changes that occur after surgery, and irritable bowelsyndrome, which comprises maintaining chronic steady state plasma levelsbetween about 60/X picomolar and about 200/X picomolar of a GLP-1 analogor derivative in a biologically active form wherein X is the in vitropotency of the GLP-1 analog or derivative relative to Val⁸-GLP-1(7-37)OHwhich is a given a value of 1 and wherein the GLP-1 analog or derivativeis administered by subcutaneous injection no more than once or twiceevery 24 hours.

[0012] The present invention also encompasses use of a GLP-1 analog orderivative having an in vitro potency within 2-fold that ofVal⁸-GLP-1(7-37)OH for the manufacture of a medicament for normalizingblood glucose, preserving β cells, inducing weight loss, or treating acondition selected from the group consisting of: hyperglycemia, type 2diabetes, stroke, myocardial infarction, catabolic changes that occurafter surgery, obesity, and irritable bowel syndrome which comprisesmaintaining chronic steady state plasma levels of the GLP-1 analog orderivative between about 60 picomolar and about 200 picomolar andwherein the GLP-1 analog or derivative is administered by subcutaneousinjection not more the once or twice every 24 hours.

[0013] The present invention also encompasses use of a GLP-1 analog orderivative for the manufacture of a medicament for normalizing bloodglucose, preserving β cells, inducing weight loss, or treating acondition selected from the group consisting of: hyperglycemia, type 2diabetes, stroke, myocardial infarction, catabolic changes that occurafter surgery, obesity, and irritable bowel syndrome which comprisesmaintaining chronic steady state plasma levels of the GLP-1 analog orderivative between about 60/X picomolar and about 200/X picomolarwherein X is the in vitro potency of the GLP-1 analog or derivativerelative to Val⁸-GLP-1(7-37)OH which is given a reference value of 1 andwherein the GLP-1 analog or derivative is administered by subcutaneousinjection no more than once or twice every 24 hours.

[0014] The invention also encompasses an article of manufacture forhuman pharmaceutical use comprising a container; a dosage formcomprising an amount of a GLP-1 analog or derivative having an in vitropotency within two-fold that of Val⁸-GLP-1(7-37)OH, and a package insertthat provides for administration of the dosage form that results inmaintaining GLP-1 analog or derivative plasma levels between about 60picomolar and about 200 picomolar.

[0015]FIG. 1: Graphs representing the mean (+/−SEM) plasmaVal⁸-GLP-1(7-37)OH concentrations following once-daily administration ofplacebo (baseline), 2.5 mg (Group 1), and 3.5 mg (Group 2) ofVal⁸-GLP-1(7-37)OH to patients with type 2 diabetes.

[0016]FIG. 2: Graphs representing the mean (+/−SEM) glucoseconcentrations following once-daily administration of placebo(baseline), 2.5 mg (Group 1), and 3.5 mg (Group 2) of Val⁸-GLP-1(7-37)OHto patients with type 2 diabetes.

[0017]FIG. 3: Graphs representing the mean (+/−SEM) plasmaVal⁸-GLP-1(7-37)OH concentrations following once-daily administration ofplacebo (baseline) and 4.5 mg (Groups 3 and 4) of Val⁸-GLP-1(7-37)OH topatients with type 2 diabetes.

[0018]FIG. 4: Graphs representing the mean (+/−SEM) glucoseconcentrations following once-daily administration of placebo (baseline)and 4.5 mg (Groups 3 and 4) of Val⁸-GLP-1(7-37)OH to patients with type2 diabetes.

[0019] For purposes of the present invention as disclosed and describedherein, the following terms and abbreviations are defined as follows.

[0020] A “treatment regimen” is the administration of a GLP-1 compoundsuch that optimum plasma levels are chronically maintained. The GLP-1compounds used for the regimen of the present invention exert theirbiological effects by acting at a receptor referred to as the GLP-1receptor. Subjects with diseases and/or conditions that respondfavorably to GLP-1 receptor stimulation or to the administration ofGLP-1 compounds can therefore be treated with the regimen of the presentinvention.

[0021] Thus, this regimen provides a variety of positive effectsincluding but not limited to treating hyperglycemia, maintaining bloodglucose control, treating type 2 diabetes, treating obesity, inducingweight loss, treating stroke, treating myocardial infarction, treatingcatabolic changes that occur after surgery or for other reasons,treating irritable bowel syndrome, preventing β-cell deterioration,inducing β-cell proliferation, stimulating insulin gene transcription,up-regulating IDX-1/PDX-1 or other growth factors, improving β-cellfunction, activating dormant β-cells, differentiating cells intoβ-cells, and/or β cell replication. Positive effects that result frommaintaining blood plasma levels within a specific range over extendedtime periods include an amelioration of the symptom(s) associated withthe disease or condition being treated, a delay in the onset of symptomsassociated with the disease or condition being treated, increasedlongevity compared with the absence of the treatment, and/or a greaterquality of life compared with the absence of the treatment. Furtherbenefits provided by the treatment regimen of the present inventionwhich relate to the treatment of type 2 diabetes and associatedhyperglycemia include enhanced convenience due to the elimination orreduction of blood glucose self-monitoring and administration of drugthat need not be timed with meals.

[0022] “Chronic therapy” refers to maintaining blood plasma levels ofactive GLP-1 compounds within a specific range for a course of therapy.The specified range corresponds to plasma levels of active GLP-1compounds that provide optimal efficacy and yet do not cause or at leastminimize side effects such as nausea and vomiting. A planned course oftherapy will differ depending on the condition or disease being treated.For example, a planned course of therapy for a type 2 diabetic whereinoral medications are no longer able to control blood glucose levelswould encompass that time period wherein the patient has adequate β cellfunction to respond to GLP-1 receptor stimulation. A planned course oftherapy for an obese patient or a patient desiring to lose weight wouldencompass that time period until the patient has reached a normal weightbased on the patient's height and build. A planned course of therapy mayalso have a prophylactic goal such as to prevent the progression of type2 diabetes, the development of diabetes, impaired glucose tolerance,syndrome x, or to prevent weight gain. This type of therapy couldpotentially last a patient's lifetime.

[0023] “Chronic” generally refers to regular administration for anextended period preferably not more frequently than twice daily, mostpreferably not more than once daily. However, chronic administration asused herein may encompass other regimens in addition to once or twicedaily dosing. For example, chronic administration encompassesadministration of a sustained release formulation that providessufficient therapeutic blood plasma levels on a regular basis. Suchadministration may include administration once a week, once a month, oreven less frequently. Contrary to acute or on-demand administration,chronic administration does not link administration of drug to eventssuch as meals, results of home glucose monitoring, or need for appetitesuppression.

[0024] “Insulinotropic activity” refers to the ability to stimulateinsulin secretion in response to elevated glucose levels, therebycausing glucose uptake by cells and decreased plasma glucose levels.Insulinotropic activity can be assessed by methods known in the art,including using in vivo experiments and in vitro assays that measureGLP-1 receptor binding activity or receptor activation, e.g., assaysemploying pancreatic islet cells or insulinoma cells, as described in EP619,322 to Gelfand, et al., and U.S. Pat. No. 5,120,712, respectively.The entire teachings of these references are incorporated herein byreference. Insulinotropic activity is routinely measured in humans bymeasuring insulin levels or C-peptide levels.

[0025] “Container” means any receptacle and closure suitable forstoring, shipping, dispensing, and/or handling a pharmaceutical product.

[0026] “Packaging” means a customer-friendly device allowing convenientadministration and/or ancillary devices that aid in delivery, education,and/or administration. The packaging may improve GLP-1 compoundadministration to the patient, reduce or improve educational instructiontime for the patient, provide a platform for improved health economicstudies, and/or limit distribution channel workload. Also, the packagingmay include but not be limited to a paper-based package, shrink wrappedpackage, see-through top packaging, trial-use coupons, educationalmaterials, ancillary supplies, and/or delivery device.

[0027] “Package insert” means information accompanying the product thatprovides a description of how to administer the product, along with thesafety and efficacy data required to allow the physician, pharmacist,and patient to make an informed decision regarding use of the product,and/or patient education information. The package insert generally isregarded as the “label” for a pharmaceutical product.

[0028] A “subject” or “patient” is a human.

[0029] “In vitro potency” as used herein is the measure of the potencyor ability of a compound to activate the GLP-1 receptor in a cell-basedassay. In vitro potency is expressed as the “EC₅₀” which is theeffective concentration of compound that results in 50% activity in asingle dose-response experiment. For the purposes of the presentinvention, in vitro potency is determined using a fluorescence assaythat employs HEK-293 Aurora CRE-BLAM cells that stably express the humanGLP-1 receptor. The assay is discussed in more detail on page 17 and inexample 3. The in vitro potency values as disclosed herein are expressedas the EC₅₀ which was established by generating a dose response curveusing dilutions resulting in GLP-1 compound concentrations from 3nanomolar to 30 nanomolar. Relative in vitro potency values areestablished by running Val⁸-GLP-1(7-37)OH as a control and assigning thecontrol a reference value of 1.

[0030] The GLP-1 compounds of the present invention have sufficienthomology to GLP-1(7-37)OH or a fragment of GLP-1(7-37)OH such that thecompound has the ability to bind to the GLP-1 receptor and initiate asignal transduction pathway resulting in insulinotropic action or otherphysiological effects as described herein such as inhibition of glucagonand delay in gastric emptying. For example, GLP-1 compounds can betested for insulinotropic activity using a cell-based assay such as thatdescribed in EP 619 322 which is a modification of the method describedby Lacy, et al. (1967) Diabetes 16:35-39. A collagenase digest ofpancreatic tissue is separated on a Ficoll gradient (27%, 23%, 20.5%,and 11% in Hank's balanced salt solution, pH 7.4). The islets arecollected from the 20.5%/11% interface, washed and handpicked free ofexocrine and other tissue under a stereomicroscope. The islets areincubated overnight in RPMI 1640 medium supplemented with 10% fetalbovine plasma and containing 11 mM glucose at 37° C. and 95% air/5% CO₂.The GLP-1 compound to be studied is prepared at a range ofconcentrations, preferably 3 nanomolar to 30 nanomolar in RPMI mediumcontaining 10% fetal bovine plasma and 16.7 mM glucose. About 8 to 10isolated islets are then transferred by pipette to a total volume of 250μl of the GLP-1 compound containing medium in 96 well microtiter dishes.The islets are incubated in the presence of the GLP-1 compound at 37°C., 95% air, 5% CO₂ for 90 minutes. Then aliquots of islet-free mediumare collected and 100 μl thereof are assayed for the amount of insulinpresent by radioimmunoassay using an Equate Insulin RIA Kit (Binax,Inc., Portland, Me.).

[0031] It is preferred that the GLP-1 compounds of the present inventionhave an in vitro potency no more than 10-fold lower than the in vitropotency of Val⁸-GLP-1(7-37)OH. Preferably, the GLP-1 compounds have anin vitro potency not lower than the in vitro potency ofVal⁸-GLP-1(7-37)OH. Representative GLP-1 compounds are discussed indetail below. Furthermore, the GLP-1 compounds used in the chronictreatment regimen described herein may require modification orformulation such that blood plasma levels are maintained in the claimedefficacious range for extended time periods. Modification andformulation of GLP-1 compounds is also discussed in detail below.

[0032] Although GLP-1 has been proposed as a possible therapy for type 2diabetes, its short half-life and susceptibility to protease degradationhas made it a difficult molecule to study. Furthermore, side effectssuch as nausea and vomiting have been observed after a singlesubcutaneous or i.v. bolus administration of active GLP-1. Applicantsbelieve this is due to the initial peak levels of the compound that areobtained immediately after administration. In order for a short actingformulation to provide a therapeutic benefit, it must be injected at ahigh enough dose to provide blood levels that are in the therapeuticrange at least long enough to achieve a glucose lowering effect after ameal. These undesired effects occurring after administration of arelatively high dose of a short-acting GLP-1 formulation limit theamount that can be administered to patients and correspondingly limitsthe efficacy.

[0033] Clinical studies have established several of the physiologicaleffects of GLP-1 which include stimulation of insulin secretion,inhibition of glucagon secretion, decrease in hepatic glucoseproduction, inhibition of gastric emptying, and promotion of weightloss. However, GLP-1 compounds cannot be effectively used in a treatmentregimen unless pharmacological levels of active GLP-1 are presentcontinuously throughout the course of treatment. This is particularlytrue in order to fully exploit blood glucose lowering potential as wellas other long-term physiological effects described herein.

[0034] Accordingly, the present invention describes the steady stateplasma levels of an active GLP-1 compound having a specific potencynecessary to achieve efficacy yet avoid or minimize side effects such asnausea and vomiting. The steady state concentration of a drug isachieved when drug elimination which is a product of clearance andconcentration equals the rate of drug availability. In the context ofintermittent dosage, during each interdose interval, the concentrationof drug rises and falls. At steady state, the entire cycle is repeatedidentically in each interval. However, as discussed herein, markedfluctuations in active GLP-1 plasma concentrations between doses isresponsible for side effects such as nausea and vomiting and do notresult in an optimal biological response.

[0035] The treatment regimen of the present invention involvesadministering a GLP-1 compound such that continuous steady state plasmalevels of the compound are maintained throughout a particular course oftreatment for a particular condition. In the context of the presentinvention, “maintaining” plasma levels means that the plasmaconcentration of drug during the course of treatment does not fluctuatesignificantly once steady state levels are achieved and thus, sideeffects such as nausea and vomiting are avoided or minimized and at thesame time an optimal therapeutic effect is obtained. Drug levels do notfluctuate significantly if they remain within the claimed efficaciousrange once steady state plasma levels are achieved. Surprisingly, it wasdiscovered that the therapeutic plasma levels for exogenouslyadministered GLP-1 compounds having a similar potency to native GLP-1 issignificantly higher than levels of endogenously secreted GLP-1 in thecirculation.

[0036] The present invention is based on data generated from a clinicaltrial wherein a long-acting GLP-1 formulation was administered viasubcutaneous injection once a day at three different dose levels. Aftersix days of dosing, drug levels reached a steady state plateau that wasmaintained continuously during the course of treatment. The chronictreatment regimen of the present invention may involve a GLP-1 compoundadministered continuously in order to obtain plasma levels within therange described herein or more preferably involves the administration ofa long-acting GLP-1 compound. Long acting in the context of the presentinvention means that the plasma levels of an active GLP-1 compound staywithin the therapeutic range described herein for at least 12 hoursafter delivery of a single dose. Preferably plasma levels remain withinthis range for at least 24 hours after delivery of a single dose. Thispreferred time action would result in once a day dosing.

[0037] Following administration of a sustained release formulationcontaining Val⁸-GLP-1(7-37)OH on day 1, mean Cmax values of 105, 147,300, and 222 pg/mL were achieved for doses corresponding to 2.5 mg, 3.5mg, and two groups at 4.5 mg, respectively. These Cmax values representthe mean maximum plasma concentration of intact Val⁸-GLP-1(7-37)OHachieved for a group of 8 patients at one of the given doses during thefirst day of treatment. (See FIGS. 1 and 3). The plasma concentration ofVal⁸-GLP-1(7-37)OH for all three groups resulted in some glucoselowering with levels above 200 pg/mL showing the most significant effect(Table 1). Inspection of the mean plasma profiles suggested that steadystate was essentially attained after once a day dosing for 6 days andthat the accumulation of drug was approximately 3-fold. On day 6, themean Cmax values for the 2.5 mg, 3.5 mg, and 4.5 mg dosage groups were534, 525, and 570 pg/mL, respectively. The corresponding AUC₍₀₋₂₄₎values which represent exposure to the active drug were also similar:8878, 9846, and 10619 ng*h/L, respectively. Thus, a 1.8-fold increase indose was associated with a 1.2-fold increase in the mean steady stateexposure AUC(0-24) (See FIGS. 1 and 3). TABLE 1 Dose (mg)/ Day 0 GroupParameter (Placebo) Day 1 Day 6 Day 21 2.5/1 R_(max) 267 (17.0) 246(19.3) 205 (16.3) — (mg/dL) AUC₍₀₋₄₎ 901 (18.8) 833 (19.2) 654 (21.2) —(mg*h/dL) 3.5/2 R_(max) 265 (15.9) 214 (23.9) 175 (14.7) — (mg/dL)AUC₍₀₋₄₎ 871 (18.2) 738 (22.2) 557 (14.3) — (mg*h/dL) 4.5/3 R_(max) 287(22.5) 244 (293) 221 (34.9) — (mg/dL) AUC₍₀₋₄₎ 995 (24.1) 834 (31.5) 704(35.0) — (mg*h/dL) 4.5/4 R_(max) 226 (15.1) 177 (22.0) 159 (20.2) 156(26.6) (mg/dL) AUC₍₀₋₄₎ 759 (14.8) 592 (20.4) 529 (21.0) 516 (33.5)(mg*h/dL)

[0038] A clinically relevant fall in the pre-dose fasting blood glucosewas seen after dosing in all treatment groups. The mean maximum observedglucose concentrations represented as Rmax ranged from 23% to 34% lowerthan the placebo controlled group on the sixth day of treatment.Furthermore, the glucose response of day 6 was similar to that seenafter 21 days of treatment (See FIGS. 2 and 4). A plateau in theresponse was achieved at steady state concentration corresponding to the2.5 mg and 3.5 mg doses which resulted in mean Cmax values of 534 and525 pg/mL, respectively. Unexpectantly, no severe nausea and vomitingand only occasional, generally short episodes of nausea or vomiting wasobserved in groups having plasma levels below 600 pg/mL. One patientreceived a dose that resulted in a Cmax of 990 pg/mL ofVal⁸-GLP-1(7-37)OH and this higher level was associated with somenausea.

[0039] In addition, weight loss occurred in the treatment groups. Theaverage amount of weight loss per patient during the 21-day dosingperiod was approximately 2.1 kg.

[0040] Because there will be differences in the molecular weight ofGLP-1 compounds having similar potencies, the observed plasma levels forVal⁸-GLP-1(7-37)OH are converted from pg/mL to picomolar (pmoles/L).Thus, the preferred range of plasma levels that provide maximum efficacyand yet avoid or minimize side effects such as nausea and vomiting isbetween about 60 and about 200 pmoles/liter for GLP-1 compounds having apotency that is similar or within two-fold the potency ofVal⁸-GLP-1(7-37)OH. More preferably, plasma levels are between about 80picomolar and about 200 picomolar. Even more preferably, plasma levelsare between about 100 picomolar and about 200 picomolar.

[0041] Thus, the invention also relates to the use of a GLP-1 compoundhaving a potency that is similar or within two-fold the potency ofVal⁸-GLP-1(7-37)OH for the manufacture of a medicament for thenormalization of blood glucose, preservation of β-cells, induction ofweight loss or the treatment of a condition selected from the groupconsisting of: hyperglycemia, type 2 diabetes, stroke, myocardialinfarction, catabolic changes that occur after surgery, obesity, andirritable bowel syndrome, wherein the medicament is adapted for chronicadministration such that chronic steady state plasma levels of the GLP-1compound are maintained between about 60 picomolar and about 200picomolar, preferably between about 80 picomolar and about 200picomolar, more preferably between about 100 picomolar and about 200picomolar

[0042] Plasma levels as discussed herein refer to the concentration ofan active GLP-1 compound as measured in blood plasma. Plasma contains anenzyme known as DPP-IV which readily cleaves amino acids at theN-terminus of GLP-1 compounds. It is known that GLP-1 must have anintact Histidine at the N-terminus to be active. For exampleGLP-1(7-37)OH is rapidly degraded to GLP-1(9-37)OH once it is releasedinto the plasma. GLP-1(9-37)OH is not active. Furthermore, GLP-1 canalso be inactivated by cleavage at the C-terminus. An inactiveGLP-1(7-33) metabolite has also been reported in the literature. Theplasma levels described herein for Val⁸-GLP-1(7-37)OH were measuredusing a sandwich radioimmunoassay. The assay makes use of an antibodythat specifically recognizes the intact amino-terminus ofVal⁸-GLP-1(7-37)OH in combination with another antibody which recognizesthe intact C-terminus of Val⁸-GLP-1(7-37)OH. Thus, only plasma levels ofactive Val⁸-GLP-1(7-37)OH are measured. (See Example 2).

[0043] Plasma levels of active GLP-1 compounds other thanVal⁸-GLP-1(7-37)OH can similarly be measured by generating antibodies bymethods well-known in the art that specifically identify the intactN-terminus of the compound being tested and do not cross-react withnative GLP-1.

[0044] Some GLP-1 derivatives such as Arg³⁴Lys²⁶-(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37) are long-acting because they bind toplasma albumin and slowly dissociate from albumin and are released intothe plasma as unbound derivatives that can bind the GLP-1 receptor andinitiate a signal. For the purposes of the present invention, plasmalevels refer to the concentration of active GLP-1 derivatives such asArg³⁴Lys²⁶-(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37) that are presentin the plasma not bound to albumin.

[0045] To achieve maximum efficacy while minimizing side effects, theplasma levels of a GLP-1 compound should not fluctuate significantlyonce steady state levels are obtained during the course of treatment.Levels do not fluctuate significantly if they are maintained within theranges described herein once steady state levels are achieved throughouta course of treatment. Most preferably, plasma levels of a GLP-1compound with a potency similar to or within two-fold that ofVal⁸-GLP-1(7-37)OH are maintained between about 100 picomolar and about200 picomolar throughout a course of treatment once steady state levelsare obtained. For example, FIG. 3 depicts plasma levels ofVal⁸-GLP-1(7-37)OH which remain flat and do not fluctuate significantlyover the course of 15 days based on once a day dosing. Levels aremaintained between about 400 and about 600 pg/mL which corresponds tobetween about 120 picomolar and 180 picomolar.

[0046] The optimal range of plasma levels appropriate forVal⁸-GLP-1(7-37)OH and GLP-1 compounds of similar potency (See Table 2)can also be applied to other GLP-1 compounds including Exendin-3 andExendin-4 which have different potencies. GLP-1 compounds of similarpotency include compounds that have within two-fold the activity ofVal⁸-GLP-1(7-37)OH as measured by an in vitro potency assay.

[0047] The preferred assay for the purposes of the present inventionmeasures EC₅₀ potency using HEK-293 Aurora CRE-BLAM cells that stablyexpress the human GLP-1 receptor. These HEK-293 cells have stablyintegrated a DNA vector having a cAMP response element (CRE) drivingexpression of the β-lactamase (BLAM) gene. The interaction of a GLP-1agonist with the receptor initiates a signal that results in activationof the cAMP response element and subsequent expression of β-lactamase.The β-lactamase CCF2/AM substrate that emits flourescence when it iscleaved by β-lactamase (Aurora Biosciences Corp.) can then be added tocells that have been exposed to a specific amount of GLP-1 agonist toprovide a measure of GLP-1 agonist potency. The assay is furtherdescribed in Zlokarnik, et al. (1998) Science 279:84-88 (See alsoExample 3). The EC₅₀ values listed in Table 2 were determined using theBLAM assay described above by generating a dose response curve usingdilutions from 3 nanomolar to 30 nanomolar.

[0048] Exendin-4 has a potency that is approximately 5-fold higher thanVal⁸-GLP-1(7-37)OH; thus, optimum plasma levels of Exendin-4 will beapproximately 5-fold lower than the levels appropriate forVal⁸-GLP-1(7-37)OH and compounds of similar potency. This wouldcorrespond to plasma levels in the range between about 6 picomolar andabout 40 picomolar, preferably between about 12 picomolar and about 30picomolar. Another example of a GLP-1 compound with increased potency isVal⁸-Glu²²-GLP-1(7-37)OH which has a potency approximately 3-fold higherthan Val⁸-GLP-1(7-37)OH. Thus, optimum plasma levels of this compoundwill be approximately 3-fold lower than the levels determined for.Val⁸-GLP-1 (7-37) OH. TABLE 2 In vitro activity relative to GLP-1Compound Val⁸-GLP-1(7-37)OH Val⁸-GLP-1(7-37)OH 1 Val⁸-GLP-1(7-36)NH₂1.06 GLP-1(7-37)OH 2.06 GLP-1(7-36)NH₂ 1.50 Gly⁸-GLP-1(7-37)OH 1.67Val⁸-Tyr¹²-GLP-1(7-37)OH 1.73 Val⁸-Trp¹²-GLP-1(7-37)OH 1.07Val⁸-Leu¹⁶-GLP-1(7-37)OH 1.13 Val⁸-Lys²²-GLP-1(7-37)OH 1.22 Exendin-44.5 Val⁸-Glu²²-GLP-1(7-37)OH 3.33 Val⁸-Arg²⁶-GLP-1(7-37)OH 1.47Val⁸-Ala²⁷-GLP-1(7-37)OH 1 Arg³⁴Lys²⁶-(N-ε-(γ-Glu(N-α- 1.92hexadecanoyl)))-GLP-1(7-37)

[0049] Thus, the range of plasma levels appropriate for a GLP-1 compoundwith a potency that differs from that of Val⁸-GLP-1(7-37)OH can bedetermined. For example, a range of plasma levels for a particular GLP-1compound is between about 60/X and 200/X, preferably between about 60/Xand 150/X, most preferably between about 100/X and about 150/X wherein Xis the in vitro potency of the GLP-1 compound relative toVal⁸-GLP-1(7-37)OH wherein Val⁸-GLP-1(7-37)OH has a reference value of1.

[0050] Further, the invention relates to the use of a GLP-1 compound forthe manufacture of a medicament for the normalization of blood glucose,preservation of β-cells, induction of weight loss, or the treatment of acondition selected from the group consisting of: hyperglycemia, type 2diabetes, stroke, myocardial infarction, catabolic changes that occurafter surgery, obesity, and irritable bowel syndrome, wherein themedicament is adapted for chronic administration such that chronicplasma levels of the GLP-1 compound are maintained between about 60/Xpicomolar and about 200/X picomolar wherein X is the in vitro potency ofthe GLP-1 compound relative to Val⁸-GLP-1(7-37)OH which has a referencevalue of 1.

[0051] Maintaining plasma levels within the range discovered by theinventors of the present invention provides numerous clinical benefitsas well as benefits from a patient convenience standpoint. There islittle or no risk of hypoglycemia to the subject when using thistreatment regimen. Additionally, this regimen minimizes invasive,planning, and/or time-consuming events. Furthermore, the regimenprovides convenience to the patient by reducing blood glucoseself-monitoring in conjunction with use. Most preferably, blood glucoseself-monitoring is reduced significantly or eliminated for subjectsusing this treatment regimen. For example, this use does not requirepatient planning before, during, or following a meal. Most preferably,subjects do not need to link use of this regimen with any glucose,calorie, or sustenance consumption event of any quantity. Furthermore,use of this invention preferably limits any dose titration needed for asubject to determine the effective amount required. Most preferably, nodose titration is required thereby making one or two doses appropriatefor all patients.

[0052] While pre-clinical data has alluded to some of the long-termhealth benefits associated with GLP-1 therapy, it has not been possibleto take advantage of these long-term benefits in human patients due tothe lack of understanding regarding the steady plasma levels required toachieve such benefits.

[0053] Maintaining plasma levels of intact GLP-1 compounds as describedherein induce long-term benefits derived from the suppression ofglucagon, upregulation of somatostatin, stimulation of insulin genetranscription, up-regulation of IDX-1/PDX-1 or other growth factors,improvement of β cell function, activation of dormant β cells,differentiation of cells into β cells, β cell replication, and β cellproliferation. For the purposes of the present invention, a method ofpreserving β cells may be due to all or some or one of the followingeffects: improvement of β cell function, activation of dormant β cells,differentiation of cells into β cells, β cell replication, preventingβ-cell deterioration such as by inhibition of apoptosis, and β cellproliferation.

[0054] Maintaining plasma levels of intact GLP-1 compounds as describedherein induce long-term benefits such as appetite suppression resultingin weight loss or lack of weight gain. For example, obesity and relatedconditions are treated or prevented by this chronic treatment regimen.Any and all reduction in weight via less weight gain, no weight gain,and/or weight loss provides the subject with overall positive physicaland psychological health effects, contributes to lessening risk factorslinked to excessive body weight, and enforces compliant use of thecompounds thereby reducing potential blood glucose excursions and itsconcomitant effects.

[0055] Another benefit of chronic exposure to GLP-1 compounds within therange of claimed serum levels includes the elimination of the delay ongastric emptying that occurs when GLP-1 compounds are firstadministered. By analyzing the timing of glucose peaks relative to theingestion of a meal for patients receiving a GLP-1 compound, it wasdetermined that the delay in gastric emptying caused by the presence ofa GLP-1 compound is approximately 2 to 3 hours. Surprisingly, after 6days of chronic GLP-1 compound therapy, the analysis of glucose peaksindicated that this delay in gastric emptying was eliminated. Thus,chronic exposure to GLP-1 compounds within the claimed serum level rangeleads to an elimination of GI effects such as a delay in gastricemptying and, therefore, increases patient tolerability to the drug andpotentially minimizes side effects.

[0056] This chronic treatment regimen may include treatment using GLP-1compounds along with other blood glucose lowering drugs such asmetformin, sulfonyl ureas, thiazolidinediones, and/or bisguanidines. Therange of plasma levels described herein is appropriate when GLP-1compounds are used as a monotherapy or used in conjunction with oralanti-diabetic agents.

[0057] The term “GLP-1 compounds” refers to GLP-1(7-37)OH andGLP-1(7-36)NH₂ and analogs and derivatives thereof. GLP-1 compounds alsoinclude Exendin-3 and Exendin-4 and analogs and derivatives thereof. Anyof these GLP-1 compounds may need further modification or be formulatedsuch that blood plasma levels are maintained for extended time periodsfollowing a single dose. GLP-1 peptides can be made by a variety ofmethods known in the art such as solid-phase synthetic chemistry,purification of GLP-1 molecules from natural sources, recombinant DNAtechnology, or a combination of these methods. For example, methods forpreparing GLP-1 peptides are described in U.S. Pat. Nos. 5,118,666,5,120,712, 5,512,549, 5,977,071, and 6,191,102. As is the custom in theart, the N-terminal residue of a GLP-1 compound is represented asposition 7.

[0058] The two naturally occurring truncated GLP-1 peptides arerepresented in formula I, SEQ ID NO: 1. SEQ ID NO:17   8   9   10  11  12  13  14  15  16  17His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-18  19  20  21  22  23  24  25  26  27  28Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-29  30  31  32  33  34  35  36  37 Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-XaaFormula I,

[0059] wherein:

[0060] Xaa at position 37 is Gly, or —NH₂.

[0061] Preferably, a GLP-1 compound has the amino acid sequence of SEQID NO. 1 or is modified so that from one, two, three, four or five aminoacids differ from SEQ ID NO: 1.

[0062] Some GLP-1 compounds known in the art include, for example,GLP-1(7-34) and GLP-1(7-35), GLP-1(7-36), Gln⁹-GLP-1(7-37),D-Gln⁹-GLP-1(7-37), Thr¹⁶-Lys¹⁸-GLP-1(7-37), and Lys¹⁸-GLP-1(7-37).GLP-1 compounds such as GLP-1(7-34) and GLP-1(7-35) are disclosed inU.S. Pat. No. 5,118,666, herein incorporated by reference. Other knownbiologically active GLP-1 analogs are disclosed in U.S. Pat. No.5,977,071; U.S. Pat. No. 5,545,618; U.S. Pat. No. 5,705,483; U.S. Pat.No. 5,977,071; U.S. Pat. No. 6,133,235: and Adelhorst, et al., J. Biol.Chem. 269:6275 (1994).

[0063] GLP-1 compounds also include polypeptides in which one or moreamino acids have been added to the N-terminus and/or C-terminus ofGLP-1(7-37)OH, or fragments or analogs thereof. Preferably from one tosix amino acids are added to the N-terminus and/or from one to eightamino acids are added to the C-terminus of GLP-1(7-37)OH. It ispreferred that GLP-1 compounds of this type have up to about thirty-nineamino acids. The amino acids in the “extended” GLP-1 compounds aredenoted by the same number as the corresponding amino acid inGLP-1(7-37)OH. For example, the N-terminal amino acid of a GLP-1compound obtained by adding two amino acids to the N-terminus ofGLP-1(7-37)OH is at position 5; and the C-terminal amino acid of a GLP-1compound obtained by adding one amino acid to the C-terminus ofGLP-1(7-37)OH is at position 39. Amino acids 1-6 of an extended GLP-1compound are preferably the same as or a conservative substitution ofthe amino acid at the corresponding position of GLP-1(1-37)OH. Aminoacids 38-45 of an extended GLP-1 compound are preferably the same as ora conservative substitution of the amino acid at the correspondingposition of Exendin-3 or Exendin-4. The amino acid sequence of Exendin-3and Exendin-4 are represented in formula II, SEQ ID NO: 2. SEQ ID NO: 27   8   9   10  12  12  13  14  15  16  17His-Xaa-Xaa-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser18  19  20  21  22  23  24  25  26  27  28Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-29  30  31  32  33  34  35  36  37  38  39Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser- 40  41  42  43  44  45Gly-Ala-Pro-Pro-Pro-Ser

[0064] wherein:

[0065] Xaa at position 8 is Ser or Gly; and

[0066] Xaa at position 9 is Asp or Glu;

[0067] As used herein, a conservative substitution is the replacement ofan amino acid with another amino acid that has the same net electroniccharge and approximately the same size and shape. Amino acids withaliphatic or substituted aliphatic amino acid side chains haveapproximately the same size when the total number carbon and heteroatomsin their side chains differs by no more than about four. They haveapproximately the same shape when the number of branches in the theirside chains differs by no more than one. Amino acids with phenyl orsubstituted phenyl groups in their side chains are considered to haveabout the same size and shape.

[0068] A preferred group of GLP-1 compounds is comprised of GLP-1analogs of formula III (SEQ ID NO: 3): (SEQ ID NO: 3)7   8   9   10  11  12  13  14  15  16  17Xaa-Xaa-Xaa-Gly-Xaa-Xaa-Thr-Xaa-Asp-Xaa-Xaa-18  19  20  21  22  23  24  25  26  27  28Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Phe-29  30  31  32  33  34  35  36  37  38  39Ile-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa-Xaa- 40  41  42  43  44  45Xaa-Xaa-Xaa-Xaa-Xaa-Xaa Formula III

[0069] wherein:

[0070] Xaa at position 7 is: L-histidine, D-histidine,desamino-histidine, 2-amino-histidine, β-hydroxy-histidine,homohistidine, α-fluoromethyl-histidine or α-methyl-histidine; Xaa atposition 8 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys; Xaaat position 9 is Glu, Asp, Lys, Thr, Ser, Arg, Trp, Phe, Tyr, or His;Xaa at position 11 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Glu, Asp, Arg,His, or Lys; Xaa at position 14 is Ser, Ala, Gly, Thr, Leu, Ile, Val,Glu, Asp, or Lys; Xaa at position 12 is His, Trp, Phe, or Tyr Xaa atposition 16 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Tyr, Glu, Asp, Trp,His, Phe, or Lys; Xaa at position 17 is Ser, Ala, Gly, Thr, Leu, Ile,Val, Glu, Asp, or Lys; Xaa at position 18 is Ser, Ala, Gly, Thr, Leu,Ile, Val, Glu, Asp, His, Pro, Arg, or Lys; Xaa at position 19 is Tyr,Phe, Trp, Glu, Asp, Gly, Gln, Asn, Arg, Cys, or Lys; Xaa at position 20is Leu, Ala, Gly, Ser, Thr, Ile, Val, Glu, Asp, Met, or Lys; Xaa atposition 21 is Glu, Asp, or Lys; Xaa at position 22 is Gly, Ala, Ser,Thr, Leu, Ile, Val, Glu, Asp, or Lys; Xaa at position 23 is Gln, Asn,Arg, Glu, Asp, His, or Lys; Xaa at position 24 is Ala, Gly, Ser, Thr,Leu, Ile, Val, Arg, Glu, Asp, or Lys; Xaa at position 25 is Ala, Gly,Ser, Thr, Leu, Ile, Val, Glu, Asp, or Lys; Xaa at position 26 is Lys,Arg, Gln, Glu, Asp, Trp, Tyr, Phe, or His; Xaa at position 27 is Glu,Asp, Ala, His, Phe, Tyr, Trp, Arg, Leu, or Lys; Xaa at position 30 isAla, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp, His, or Lys; Xaa atposition 31 is Trp, Phe, Tyr, Glu, Asp, Ser, Thr, Arg, or Lys; Xaa atposition 32 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp, or Lys; Xaaat position 33 is Val, Gly, Ala, Ser, Thr, Leu, Ile, Glu, Asp, Arg, orLys; Xaa at position 34 is Lys, Arg, Glu, Asp, Asn, or His; Xaa atposition 35 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Glu, Asp, Arg, Trp,Tyr, Phe, Pro, His, or Lys; Xaa at position 36 is Arg, Lys, Glu, Asp,Thr, Ser, Trp, Tyr, Phe, Gly, or His; Xaa at position 37 is Gly, Ala,Ser, Thr, Leu, Ile, Val, Glu, Asp, His, Lys, Arg, Trp, Tyr, Phe,Gly-Pro, Gly- Pro-NH₂, —NH₂ or is deleted; Xaa at position 38 is Arg,Lys, Glu, Asp, Ser, or His, or is deleted; Xaa at position 39 is Arg,Lys, Glu, Asp, Ser, or His, or is deleted; Xaa at position 40 is Asp,Glu, Gly, or Lys, or is deleted; Xaa at position 41 is Phe, Trp, Tyr,Glu, Asp, Ala, or Lys, or is deleted; Xaa at position 42 is Pro, Lye,Glu, or Asp, or is deleted; Xaa at position 43 is Glu, Asp, Pro, or Lys,or is deleted; Xaa at position 44 is Glu, Asp, Pro, or Lys, or isdeleted; and Xaa at position 45 is Val, Glu, Asp, Ser, or Lys, or isdeleted, or

[0071] a C-1-6-ester, or amide, or C-1-6-alkylamide, orC-1-6-dialkylamide thereof; provided that when the amino acid atposition 37, 38, 39, 40, 41, 42, 43, or 44 is deleted, then each aminoacid downstream of that amino acid is also deleted. It is preferred thatthe analogs encompassed by formula III, have not more than six aminoacids that differ from the corresponding amino acids in GLP-1(7-37)OH,GLP-1(7-36)NH₂, Exendin-3, or Exendin-4. It is more preferred that theanalogs encompassed by formula III have between one and five amino acidsthat differ from the corresponding amino acids in GLP-1(7-37)OH,GLP-1(7-36)NH₂, Exendin-3, or Exendin-4.

[0072] Another preferred group of GLP-1 compounds is comprised of GLP-1analogs of formula IV (SEQ ID NO: 4):His-Xaa₈-Glu-Gly-Xaa₁₁-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser- (SEQ ID NO: 4)Ser-Tyr-Leu-Glu-Xaa₂₂-Xaa₂₃-Xaa₂₄-Ala-Xaa₂₆-Xaa₂₇-Phe-Ile-Ala-Xaa₃₁-Leu-Xaa₃₃-Xaa₃₄-Xaa₃₅-Xaa₃₆-R formula IV wherein: Xaa₈ is:Gly, Ala, Val, Leu, Ile, Ser, or Thr; Xaa₁₁ is: Asp, Glu, Arg, Thr, Ala,Lys, or His; Xaa₁₂ is: His, Trp, Phe, or Tyr; Xaa₁₆ is: Leu, Ser, Thr,Trp, His, Phe, Asp, Val, Glu, or Ala; Xaa₂₂ is: Gly, Asp, Glu, Gln, Asn,Lys, Arg, Cys, or Cysteic Acid; Xaa₂₃ is: His, Asp, Lys, Glu, or Gln;Xaa₂₄ is: Glu, His, Ala, or Lys; Xaa₂₆ is: Asp, Lys, Glu, or His; Xaa₂₇is: Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys; Xaa₃₀ is: Ala, Glu, Asp,Ser, or His; Xaa₃₃ is: Asp, Arg, Val, Lys, Ala, Gly, or Glu; Xaa₃₄ is:Glu, Lys, or Asp; Xaa₃₅ is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly,Pro, His, or Glu; Xaa₃₆ is: Arg, Glu, or His; R is: Lys, Arg, Thr, Ser,Glu, Asp, Trp, Tyr, Phe, His, —NH₂, Gly, Gly-Pro, or Gly-Pro-NH₂,

[0073] preferred that the analogs encompassed by formula IV, have notmore than six amino acids that differ from the corresponding amino acidsin GLP-1(7-37)OH, or GLP-1(7-36)NH₂. It is more preferred that theanalogs encompassed by formula IV have between one and five amino acidsthat differ from the corresponding amino acids in GLP-1(7-37)OH, orGLP-1(7-36)NH₂.

[0074] Another preferred group of GLP-1 compounds is comprised of GLP-1analogs of formula V (SEQ ID NO: 5):His-Xaa₈-Glu-Gly-Thr-Xaa₁₂-Thr-Ser-Asp-Xaa₁₆-Ser- (SEQ ID NO: 5)Ser-Tyr-Leu-Glu-Xaa₂₂-Xaa₂₃-Ala-Ala-Xaa₂₆-Glu-Phe-Ile-Xaa₃₀-Trp-Leu-Val-Lys-Xaa₃₅-Arg-R formula III wherein: Xaa₈ is: Gly,Ala, Val, Leu, Ile, Ser, or Thr; Xaa₁₂ is: His, Trp, Phe, or Tyr; Xaa₁₆is: Leu, Ser, Thr, Trp, His, Phe, Asp, Val, Glu, or Ala; Xaa₂₂ is: Gly,Asp, Glu, Gln, Asn, Lys, Arg, Cys, or Cysteic Acid; Xaa₂₃ is: His, Asp,Lys, Glu, or Gln; Xaa₂₆ is: Asp, Lys, Glu, or His; Xaa₃₀ is: Ala, Glu,Asp, Ser, or His; Xaa₃₅ is: Thr, Ser, Lys, Arg, Trp, Tyr, Phe, Asp, Gly,Pro, His, or Glu; R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe,His, —NH₂, Gly, Gly-Pro, or Gly-Pro-NH₂, or is deleted.

[0075] It is preferred that the analogs encompassed by formula V, havenot more than six amino acids that differ from the corresponding aminoacids in GLP-1(7-37)OH, or GLP-1(7-36)NH₂. It is more preferred that theanalogs encompassed by formula V have between one and five amino acidsthat differ from the corresponding amino acids in GLP-1(7-37)OH orGLP-1(7-36)NH₂.

[0076] Another preferred group of GLP-1 compounds is comprised of GLP-1analogs of formula VI (SEQ ID NO: 6):His-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser- (SEQ ID NO: 6)Tyr-Leu-Glu-Xaa₂₂-Xaa₂₃-Ala-Ala-Lys-Xaa₂₇-Phe-Ile-Xaa₃₀-Trp-Leu-Val-Lys-Gly-Arg-R formula VI wherein: Xaa₈ is: Gly, Ala,Val, Leu, Ile, Ser, or Thr; Xaa₂₂ is: Gly, Asp, Glu, Gln, Asn, Lys, Arg,Cys, or Cysteic Acid; Xaa₂₃ is: His, Asp, Lys, Glu, or Gln; Xaa₂₇ is:Ala, Glu, His, Phe, Tyr, Trp, Arg, or Lys Xaa₃₀ is: Ala, Glu, Asp, Ser,or His; R is: Lys, Arg, Thr, Ser, Glu, Asp, Trp, Tyr, Phe, His, —NH₂,Gly, Gly-Pro, or Gly-Pro-NH₂, or is deleted.

[0077] Another preferred group of GLP-1 compounds is comprised of GLP-1analogs of formula VII (SEQ ID NO. 7): (SEQ ID NO: 7)Xaa₇-Xaa₈-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Xaa₂₂-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Lys-Gly-Arg-R

[0078] wherein:

[0079] Xaa₇ is L-histidine, D-histidine, desamino-histidine,2amino-histidine, β-hydroxy-histidine, homohistidine,α-fluoromethyl-histidine or α-methyl-histidine;

[0080] Xaa₈ is glycine, alanine, valine, leucine, isoleucine, serine orthreonine. Preferably, Xaa₈ is glycine, valine, leucine, isoleucine,serine or threonine;

[0081] Xaa₂₂ is aspartic acid, glutamic acid, glutamine, asparagine,lysine, arginine, cysteine, or cysteic acid.

[0082] R is —NH₂ or Gly(OH).

[0083] Most preferred GLP-1 compounds of formula I, II, III, IV, V, andVI comprise GLP-1 analogs wherein the backbone for such analogs orfragments contains an amino acid other than alanine at position 8(position 8 analogs). Preferred amino acids at position 8 are glycine,valine, leucine, isoleucine, serine, threonine, or methionine and morepreferably are valine or glycine.

[0084] Other preferred GLP-1 compounds are GLP-1 analogs that have thesequence of GLP-1(7-37)OH except that the amino acid at position 8 ispreferably glycine, valine, leucine, isoleucine, serine, threonine, ormethionine and more preferably valine or glycine and position 22 isglutamic acid, lysine, aspartic acid, or arginine and more preferablyglutamic acid or lysine.

[0085] Other preferred GLP-1 compounds are GLP-1 analogs that have thesequence of GLP-1(7-37)OH except that the amino acid at position 8 ispreferably glycine, valine, leucine, isoleucine, serine, threonine, ormethionine and more preferably valine or glycine and position 30 isglutamic acid, aspartic acid, serine, or histidine and more preferablyglutamic acid.

[0086] Other preferred GLP-1 compounds are GLP-1 analogs that have thesequence of GLP-1(7-37)OH except that the amino acid at position 8 ispreferably glycine, valine, leucine, isoleucine, serine, threonine, ormethionine and more preferably valine or glycine and position 37 ishistidine, lysine, arginine, threonine, serine, glutamic acid, asparticacid, tryptophan, tyrosine, phenylalanine and more preferably histidine.

[0087] Other preferred GLP-1 compounds are GLP-1 analogs that have thesequence of GLP-1(7-37)OH except that the amino acid at position 8 ispreferably glycine, valine, leucine, isoleucine, serine, threonine, ormethionine and more preferably valine or glycine and position 22 isglutamic acid, lysine, aspartic acid, or arginine and more preferablyglutamic acid or lysine and position 23 is lysine, arginine, glutamicacid, aspartic acid, and histidine and more preferably lysine orglutamic acid.

[0088] Other preferred GLP-1 compounds are GLP-1 analogs that have thesequence of GLP-1(7-37)OH except that the amino acid at position 8 ispreferably glycine, valine, leucine, isoleucine, serine, threonine, ormethionine and more preferably valine or glycine and position 22 isglutamic acid, lysine, aspartic acid, or arginine and more preferablyglutamic acid or lysine and position 27 is alanine, lysine, arginine,tryptophan, tyrosine, phenylalanine, or histidine and more preferablyalanine.

[0089] In the nomenclature used herein to describe GLP-1 compounds, thesubstituting amino acid and its position is indicated prior to theparent structure. For example Val⁸-GLP-1(7-37)OH designates a GLP-1compound in which the alanine normally found at position 8 inGLP-1(7-37)OH (formula I, SEQ ID NO:1) is replaced with valine.

[0090] Other preferred GLP-1 compounds include: Val⁸-GLP-1(7-37)OH,Gly⁸-GLP-1(7-37)OH, Glu²²-GLP-1(7-37)OH, Asp²²-GLP-1(7-37)OH,Arg²²-GLP-1(7-37)OH, Lys²²-GLP-1(7-37)OH, Cys²²-GLP-1(7-37)OH,Val⁸-Glu²²-GLP-1(7-37)OH, Val⁸-Asp²²-GLP-1(7-37)OH,Val⁸-Arg²²-GLP-1(7-37)OH, Val⁸-Lys²²-GLP-1(7-37)OH,Val⁸-Cys²²-GLP-1(7-37)OH, Gly⁸-Glu²²-GLP-1(7-37)OH,Gly⁸-Asp²²-GLP-1(7-37)OH, Gly⁸-Arg²²-GLP-1(7-37)OH,Gly⁸-Lys²²-GLP-1(7-37)OH, Gly⁸-Cya²²-GLP-1(7-37)OH,Glu²²-GLP-1(7-36)NH₂, Asp²²-GLP-1(7-36)NH₂, Arg²²-GLP-1(7-36)NH₂,Lys²²-GLP-1(7-36)NH₂, Cys²²-GLP-1(7-36)NH₂, Val⁸-Glu²²-GLP-1(7-36)NH₂,Val⁸-Asp²²-GLP-1(7-36)NH₂, Val⁸-Arg²²-GLP-1(7-36)NH₂,Val⁸-Lys²²-GLP-1(7-36)NH₂, Val⁸-Cys²²-GLP-1(7-36)NH₂,Gly⁸-Glu²²-GLP-1(7-36)NH₂, Gly⁸-Asp²²-GLP-1(7-36)NH₂,Gly⁸-Arg²²-GLP-1(7-36)NH₂, Gly⁸-Lys²²-GLP-1(7-36)NH₂,Gly⁸-Cys²²-GLP-1(7-36)NH₂, Lys²³-GLP-1(7-37)OH,Val⁸-Lys²³-GLP-1(7-37)OH, Gly⁸-Lys²³-GLP-1(7-37)OH, His²⁴-GLP-1(7-37)OH,Val⁸-His²⁴-GLP-1(7-37)OH, Gly⁸-His²⁴-GLP-1(7-37)OH, Lys²⁴-GLP-1(7-37)OH,Val⁸-Lys²⁴-GLP-1(7-37)OH, Gly⁸-Lys²³-GLP-1(7-37)OH, Glu³⁰-GLP-1(7-37)OH,Val⁸-Glu³⁰-GLP-1(7-37)OH, Gly⁸-Glu³⁰-GLP-1(7-37)OH, Asp³⁰-GLP-1(7-37)OH,Val⁸-Asp³⁰-GLP-1(7-37)OH, Gly⁸-Asp³⁰-GLP-1(7-37)OH, Gln³⁰-GLP-1(7-37)OH,Val⁸-Gln³⁰-GLP-1(7-37)OH, Gly⁸-Gln³⁰-GLP-1(7-37)OH, Tyr³⁰-GLP-1(7-37)OH,Val⁸-Tyr³⁰-GLP-1(7-37)OH, Gly⁸-Tyr³⁰-GLP-1(7-37)OH, Ser³⁰-GLP-1(7-37)OH,Val⁸-Ser³⁰-GLP-1(7-37)OH, Gly⁸-Ser³⁰-GLP-1(7-37)OH, His³⁰-GLP-1(7-37)OH,Val⁸-His³⁰-GLP-1(7-37)OH, Gly⁸-His³⁰-GLP-1(7-37)OH, Glu³⁴-GLP-1(7-37)OH,Val⁸-Glu³⁴-GLP-1(7-37)OH, Gly⁸-Glu³⁴-GLP-1(7-37)OH, Ala³⁴-GLP-1(7-37)OH,Val⁸-Ala³⁴-GLP-1(7-37)OH, Gly⁸-Ala³⁴-GLP-1(7-37)OH, Gly³⁴-GLP-1(7-37)OH,Val⁸-Gly³⁴-GLP-1(7-37)OH, Gly⁸-Gly³⁴-GLP-1(7-37)OH, Ala³⁵-GLP-1(7-37)OH,Val⁸-Ala³⁵-GLP-1(7-37)OH, Gly⁸-Ala³⁵-GLP-1(7-37)OH, Lys³⁵-GLP-1(7-37)OH,Val⁸-Lys³⁵-GLP-1(7-37)OH, Gly⁸-Lys³⁵-GLP-1(7-37)OH, His³⁵-GLP-1(7-37)OHVal⁸-His³⁵-GLP-1(7-37)OH, Gly⁸-His³⁵-GLP-1(7-37)OH, Pro³⁵-GLP-1(7-37)OH,Val⁸-Pro³⁵-GLP-1(7-37)OH, Gly⁸-Pro³⁵-GLP-1(7-37)OH, Glu³⁵-GLP-1(7-37)OHVal⁸-Glu³⁵-GLP-1(7-37)OH, Gly⁸-Glu³⁵-GLP-1(7-37)OH,Val⁸-Ala²⁷-GLP-1(7-37)OH, Val⁸-His³⁷-GLP-1(7-37)OH,Val⁸-Glu²²-Lys²³-GLP-1(7-37)OH, Val⁸-Glu²²-Glu²³-GLP-1(7-37)OH,Val⁸-Glu²²-Ala²⁷-GLP-1(7-37)OH, Val⁸-Gly³⁴-Lys³⁵-GLP-1(7-37)OH,Val⁸-His³⁷-GLP-1(7-37)OH, and Gly⁸-His³⁷-GLP-1(7-37)OH.

[0091] More preferred GLP-1 compounds are Val⁸-GLP-1(7-37)OH,Gly⁸-GLP-1(7-37)OH, Glu²²-GLP-1(7-37)OH, Lys²²-GLP-1(7-37)OH,Val⁸-Glu²²-GLP-1(7-37)OH, Val⁸-Lys²²-GLP-1(7-37)OH,Gly⁸-Glu²²-GLP-1(7-37)OH, Gly⁸-Lys²²-GLP-1(7-37)OH,Glu²²-GLP-1(7-36)NH₂, Lys²²-GLP-1(7-36)NH₂, Val⁸-Glu²²-GLP-1(7-36)NH₂,Val⁸-Lys²²-GLP-1(7-36)NH₂, Gly⁸-Glu²²-GLP-1(7-36)NH₂,Gly⁸-Lys²²-GLP-1(7-36)NH₂, Val⁸-His³⁷-GLP-1(7-37)OH,Gly⁸-His³⁷-GLP-1(7-37)OH, Arg³⁴-GLP-1(7-36)NH₂, and Arg³⁴-GLP-1(7-37)OH.

[0092] A GLP-1 compound also includes a “GLP-1 derivative” which isdefined as a molecule having the amino acid sequence of GLP-1 or of aGLP-1 analog, but additionally having chemical modification of one ormore of its amino acid side groups, α-carbon atoms, terminal aminogroup, or terminal carboxylic acid group. A chemical modificationincludes, but is not limited to, adding chemical moieties, creating newbonds, and removing chemical moieties.

[0093] The GLP-1 compound used for the chronic treatment regimen mayrequire modification or formulation so that blood plasma levels aremaintained in the claimed efficacious range for extended time periods.Various means can be employed to achieve a protracted time actionincluding, for example, the incorporation of GLP-1 compounds intosuspended amorphous or crystalline particles wherein the GLP-1 compoundis complexed with zinc or protamine and slowly solubilizes uponadministration. Another means includes derivatizing a GLP-1 compoundsuch that it binds plasma albumin and slowly dissociates over time. Inaddition, depot formulations wherein an bioadsorbable polymer is used toprovide sustained release over time are also suitable for use in thepresent invention.

[0094] For example, GLP-1 compound can be incorporated into zinccrystals which have a protracted time action by dissolving the selectedGLP-1 peptide in a glycine-free solution at a pH of about 9.5 to about11.5. This “alkaline normalization” step appears to reduce the contentof β-sheet conformation in the peptide and enhance the α-helixconformation that is important for solubility and bioavailability ofsome GLP-1 compounds. This step also serves to maintain the peptide in apreferred α-helix conformation prior to the subsequent process step.This key step thus “normalizes” variation in bulk lots of the peptideinto a more reproducible, homogenous solution.

[0095] Preferably, the peptide concentration in the alkalinenormalization solution is greater than 5 mg/mL. More preferably, theconcentration is about 10 mg/mL to about 30 mg/mL. Other ranges ofpreferred concentration of dissolved peptide are about 5 mg/mL to about25 mg/mL, about 8 mg/mL to about 25 mg/mL and about 10 mg/mL to about 20mg/mL. The most preferred concentration is about 15 mg/mL.

[0096] Preferably, an aqueous alkaline solution comprising only waterand a base such as NaOH, KOH or ammonium hydroxide is employed todissolve the peptide. A more preferred base is NaOH.

[0097] Preferably, the pH of the alkaline normalization step is about10.0 to about 11.0. More preferably, the pH is about 10.5. The alkalinesolution comprising the dissolved peptide may be allowed to sitquiescently for a period of about 5 minutes to about 3 hours at ambienttemperature, which, although it is not to be construed as a limitation,is generally between about 20° C. and about 25° C. The alkaline solutionmay also be gently stirred. More preferably, the dissolved alkalinepeptide solution will sit quiescently for about 1 hour at ambienttemperature. One skilled in the art will recognize that combinations ofpH, time, temperature and stirring conditions for this step can bereadily established for each peptide that ensures “normalization” of thepeptide conformation is complete yet avoids or minimizes chemicaldegradation that may occur to the peptide.

[0098] The next step in the process for preparing crystals of a selectedpeptide is the addition of glycine. Amino acids such as glycine bindzinc ions which also bind very tightly to the histidine residue(s) in apeptide. Thus, competition for zinc binding may play a role in theformation of peptide crystals, as well as in the stability of subsequentcrystalline compositions. The glycine added to the alkaline peptidesolution may be in a solid form or in a stock solution. Preferably,glycine is added as a solid. Preferably, the added glycine is infree-base form. Preferably, the resulting concentration of glycine inthe alkaline peptide solution is about 5 mM to about 250 mM. Ranges ofmore preferred glycine concentration are about 10 mM to about 150 mM,about 20 mM to about 100 mM, about 40 mM to about 80 mM and about 55 mMto about 65 mM. Most preferably, the glycine concentration is about 60mM.

[0099] Optionally, the pH of the alkaline peptide solution may bereadjusted after the addition of the glycine. If the pH is adjusted, itis preferably adjusted to a pH between about 9.0 and about 11.0. Morepreferably, it is adjusted to a pH between about 9.2 and about 9.8. Mostpreferably, it is adjusted to about pH 9.5.

[0100] Optionally, the alkaline peptide solution with added glycine maybe filtered. Filtration is recommended if any evidence of undissolvedparticles, dust or lint is apparent in the solution. If desired, this isalso a good place in the process at which the solution can be sterilizedby performing an aseptic filtration step. Preferably, the filtrationwill be conducted using a sterile non-pyrogenic filter havinglow-protein binding and a pore size of 0.45 μm or less. Preferably, thefilter is a sterile non-pyrogenic, low-protein binding filter of poresize 0.22 μm or less. More preferably, the filter is a sterile 0.22 μmMillex® filter (Millipore Corporation, Waltham, Mass., USA).

[0101] The next step in the process of forming crystals is addition tothe alkaline peptide solution of about 2% to about 20% of the totalfinal volume of an alcohol selected from the group consisting of ethanoland isopropanol, and about 0.5 moles to about 2.5 moles of zinc per moleof the peptide. The zinc and ethanol may be added in a single aqueousstock solution or may be added separately in one or more steps in anyorder. Preferably, the alcohol is added before the zinc is added.

[0102] Preferably, the added alcohol represents, by volume, about 2% toabout 20% of the total final volume of the alkaline peptide-zinc-alcoholsolution. More preferably, the alcohol represents about 5% to about 15%of the total final volume. More preferably, the alcohol represents about6% to about 12% of the total final volume. Most preferably, the alcoholrepresents about 9% of the total final volume.

[0103] Preferably, the alcohol is ethanol.

[0104] The zinc added at this stage refers to the zinc ion. The zinc maybe added in a variety of forms, but a zinc oxide solution acidified withdilute HCl and salt forms such as zinc acetate or zinc chloride arepreferred. More preferred is a zinc oxide solution acidified with diluteHCl.

[0105] Preferably, 1.0 moles to about 2.25 moles of zinc per mole of thepeptide is added in this process step. Other preferred ranges of zincaddition include 1.1 to 2.0 moles of zinc per mole of the peptide, 1.3to 1.7 moles per mole of peptide, and 1.4 to 1.6 moles per mole ofpeptide. Most preferably, about 1.5 moles of zinc per mole of peptide isadded.

[0106] Preferably, the solution comprising zinc that is added to thepeptide solution is added slowly and/or in small increments, whichminimizes the localized precipitation of peptide and/or zinc complexesthat may form at the site of addition. More preferably, glycine is alsoa component of the solution comprising zinc that is being added at thisstep. For example, a zinc-glycine solution may be prepared by dissolvingzinc oxide in dilute HCl to a pH of about 1.6 and then adding solidglycine. A sufficient quantity of glycine is added to raise the pH ofthe solution to between about pH 2 and about pH 3. The pH of thezinc-glycine solution may be raised further using, for example, diluteNaOH. A preferred pH range of the zinc-glycine solution is about pH 4.0to about pH 6.0. A more preferred pH range of the zinc-glycine solutionis about pH 5.0 to about pH 5.5. As noted earlier, glycine has a bindingaffinity for zinc that may compete with zinc binding to the peptide.Thus, the presence of glycine in the solution comprising zinc that isbeing added to the composition allows the zinc solution to be added morequickly because localized precipitation problems are minimized. Inaddition, having a zinc-glycine solution above pH 2.0, and preferablyabout pH 4.0 to about pH 6.0, allows the solution to be sterile filteredusing filters that are rated by their manufacturers to handle, forexample, pH 2-10 solutions, prior to its introduction into a sterilepeptide composition. Preferably, the zinc-glycine solution comprisesabout 50 mM to about 70 mM glycine and about 20 mM to about 200 mM zinc.

[0107] The last steps in the initial crystallization of a selectedpeptide are adjusting the pH of the solution to between about pH 7.5 andabout pH 10.5 and allowing crystals of the peptide to form. Preferredreagent solutions useful for adjusting the pH of the solution includedilute HCl, dilute acetic acid and dilute NaOH.

[0108] Preferred pH ranges for crystallization of selected peptidesinclude about pH 8.0 to about pH 10.0, about pH 7.5 to about pH 9.5,about pH 8.5 to about pH 9.2, about pH 9.0 to about pH 9.5, about pH 7.5to about pH 8.5, about pH 8.7 to about pH 9.5, and about pH 9.2 to aboutpH 10.0.

[0109] One skilled in the art will recognize that the preferred pH ofcrystallization will depend on many factors, including the nature of thepeptide and its concentration, the alcohol concentration, the zincconcentration, the ionic strength of the solution and the temperature ofcrystallization. By way of illustration, the peptideVal⁸-Glu³⁰-GLP-1(7-37)OH produced crystals at only select ethanol andzinc concentrations at a pH range of about 7.7 to about 8.1, whereas thepeptide Val⁸-His³⁷-GLP-1(7-37)OH produced crystals over a broad range ofzinc and ethanol concentrations at a pH range of about 9.8 to about10.4.

[0110] The skilled artisan will further recognize that, for a given setof conditions, a preferred manner of determining the optimal pH ofcrystallization is to determine it empirically, that is, to slowly addthe acidification solution, preferably dilute HCl or dilute acetic acid,in small increments, and observe what happens after each increment isadded. Generally, small quantities of localized zones of precipitationwill occur at the spot of addition of the acidic solution. When gentleswirling takes increasingly longer periods of time to completelyredissolve the precipitation, that is the best time to stop adding theacid and allow crystallization from the clear or slightly cloudysolution to proceed.

[0111] The skilled artisan will further recognize that the pH andtemperature that one selects for crystallization will have an impact onthe speed at which the crystallization proceeds, the crystallizationyield, and the size and homogeneity of the crystals formed. Preferably,the pH of crystallization for the selected peptides is about pH 8.0 toabout pH 10. More preferably, the pH is about 8.7 to about 9.5. Otherranges of preferred pH of crystallization are about 8.8 to about 9.3,about 9.0 to about 9.5, and about 8.5 to about 9.3. Most preferably, thecrystallization is conducted at about pH 9.1.

[0112] Preferably, the temperature of crystallization is about 10° C. toabout 30° C. More preferably, the temperature of crystallization isabout 15° C. to about 28° C. Most preferably, the temperature ofcrystallization is ambient temperature, or about 20° C. to about 25° C.

[0113] Preferably, the crystallization step described above is complete,that is, 90% or more of the peptide is precipitated in predominantlycrystalline form, in about 3 hours to about 72 hours. More preferably,the crystallization is complete in about 10 hours to about 48 hours.Most preferably, the crystallization is complete in about 16 hours toabout 26 hours. Completion of crystallization may be determined by avariety of means, including HPLC analysis of the peptide present in analiquot of the composition. Method 5 herein describes one such protocolthat may be employed.

[0114] Preferably, the crystals produced according to the steps of theprocess described above are thin plate crystals. The crystals producedby the process may be examined by microscopy.

[0115] Pharmaceutical compositions comprising crystals of a GLP-1peptide prepared as described above may be prepared by adding suitable,pharmaceutically acceptable excipients to the crystal suspension in theoriginal mother liquor. Alternatively, the crystals may be isolated byfiltration, gentle centrifugation or other means of phase separation,and used in a variety of ways to prepare pharmaceutically acceptablecompositions. The skilled artisan will recognize suitable procedures andexcipients useful for preparing such pharmaceutical compositions.

[0116] The following process starts with the crystals and originalmother liquor from the initial crystallization stage and continues withthe preparation of a stable pharmaceutical composition.

[0117] To prepare a stable pharmaceutical composition of crystals of aselected peptide, the pH of the suspension of crystals in their completeoriginal mother liquor, or portion thereof, is lowered to a pH value atwhich 97% or more of the peptide becomes insoluble. Preferably, thispart of the process begins within a few hours after the initialcrystallization is determined to be complete. Preferably, the pH islowered using a dilute solution of HCl or acetic acid wherein the acidicsolution is added slowly and in incremental portions. The skilledartisan will recognize that the preferred pH at which this second stageof crystallization should occur will depend on many factors, includingthe nature of the peptide and its concentration, the alcoholconcentration, the zinc concentration, the ionic strength of thesuspension and the temperature of crystallization. Preferably, the pH isabout 0.2 to 2.0 pH units lower than the pH at which the initialcrystallization proceeded. More preferably, the pH is about 0.5 to about1.5 pH units lower, and most preferably, the pH is about 0.8 to 1.3 pHunits lower than the pH at which the initial crystallization proceeded.The temperature of this second stage of crystallization is preferablyambient temperature, or about 20° C. to about 25° C. For the peptideVal⁸-GLP-1(7-37)OH, a preferred pH is about 7.5 to about 8.5. A morepreferred pH is about 7.8 to about 8.2.

[0118] Preferably, the pH of a suspension of peptide crystals is loweredto a pH at which 98% or more, and more preferably at which 99% or moreof the peptide becomes insoluble in the composition. The additionalprecipitation formed in this second stage of crystallization comprisescrystals. Preferably, the additional precipitation formed in this secondstage of crystallization will be predominantly crystals of comparablemorphology and size distribution as those formed in the first stage ofcrystallization.

[0119] Preferably, the second stage of crystallization is completeenough, that is, 97% or more of the peptide is insoluble, to allow thefollowing step to begin within 30 hours, more preferably within 18hours, more preferably within 6 hours and most preferably within 2 hoursof when the second stage of crystallization started. Quantitation ofprecipitation yield may be determined by a variety of means, includingHPLC analysis of the peptide present in an aliquot of the composition.

[0120] The next step in the process to prepare a stable pharmaceuticalcomposition of crystals of a selected peptide is to add apharmaceutically acceptable preservative and a buffer selected from thegroup consisting of TRIS, maleate, phosphate, succinate, glycylglycineand adipate. Optionally, one or more tonicity agents such as sodiumchloride, other salts, glycerin or mannitol may also be added. Thesecomponents may be added as a single solution, as combination solutionsor individually in any order. It is preferred that the preservative isadded last. Of these components, a preferred buffer is selected from thegroup consisting of TRIS and maleate, a preferred preservative ism-cresol and a preferred tonicity agent is sodium chloride. A morepreferred buffer is TRIS.

[0121] A preferred quantity of TRIS to add to the crystalline peptidesuspension, if TRIS is the selected buffer, is such that the TRISconcentration in the final composition is about 5 mM to about 40 mM. Amore preferred range of TRIS concentration in the final composition isabout 10 mM to about 20 mM. A most preferred concentration of TRIS inthe final composition is about 15 mM.

[0122] A preferred quantity of maleate to add to the crystalline peptidesuspension, if maleate is the selected buffer, is such that the maleateconcentration in the final composition is about 2 mM to about 20 mM. Amore preferred range of maleate concentration in the final compositionis about 5 mM to about 15 mM. A most preferred concentration of maleatein the final composition is about 9 mM.

[0123] If sodium chloride is selected to be a component of the peptidecomposition, a preferred quantity to add to the crystalline peptidesuspension is such that the added sodium chloride in the finalcomposition is about 30 mM to about 200 mM. A more preferredconcentration of added sodium chloride in the final composition is 50 mMto about 150 mM. Other ranges of preferred sodium chloride concentrationare about 80 mM to about 120 mM, about 70 mM to about 130 mM, and about90 mM to about 130 mM. A most preferred quantity of added sodiumchloride in a pharmaceutical composition is about 110 mM.

[0124] Although any pharmaceutically acceptable preservative may beadded to the crystalline peptide suspension at this point in theprocess, for a composition of the present invention a phenolicpreservative or benzyl alcohol is preferred. Examples of phenolicpreservatives include phenol, chlorocresol, m-cresol, o-cresol,p-cresol, ethylparaben, methylparaben, propylparaben, butylparaben,thymol or mixtures thereof. More preferred preservatives are benzylalcohol, m-cresol, phenol, methylparaben and mixtures thereof. A mostpreferred pharmaceutically acceptable preservative is m-cresol.

[0125] A preferred quantity of a pharmaceutically acceptablepreservative to add to a crystalline peptide composition at this pointin the process is an amount such that the preservative concentration inthe final composition is about 1.0 mg/mL to about 20.0 mg/mL. Morepreferred ranges of concentration of preservative in the finalcomposition are about 2.0 mg/mL to about 8.0 mg/mL, about 2.5 mg/mL toabout 4.5 mg/mL and about 2.0 mg/mL to about 4.0 mg/mL. A most preferredconcentration of preservative in the final composition is about 3.0mg/mL.

[0126] The final step in the process of preparing a stablepharmaceutical composition of crystals of a selected peptide is anadjustment to a final pH between about 6.0 and about 8.5 and preferablybetween about pH 6.5 and about pH 8.0. Although any of a wide variety ofacidification and/or alkalization reagent solutions may be employed forthis pH adjustment, dilute HCl, dilute NaOH and dilute acetic acid arepreferred. More preferred reagent solutions are dilute HCl and diluteNaOH. The preferred pH to which the composition is adjusted will dependto some extent upon the selected peptide, the peptide concentration, theproposed route of administration and the selected buffer.

[0127] Preferably, with TRIS as the selected buffer, the pH will beadjusted to a pH between about 6.5 and about 8.5. More preferably, thepH will be adjusted to a pH between about 7.0 and about 7.8, betweenabout 7.2 and about 7.8, between about 7.5 and about 8.5, or betweenabout 7.0 and about 8.0. A most preferred pH to which the composition isadjusted when TRIS is the selected buffer is about 7.5. With maleate asthe selected buffer, the pH will be adjusted to a pH between about 6.0and about 7.5. More preferably, the pH will be adjusted to a pH betweenabout 6.4 and about 7.5, between about 6.4 and about 7.0, or betweenabout 6.0 and about 7.0. A most preferred pH to which the composition isadjusted when maleate is the selected buffer is about 6.5.

[0128] Instead of a formulation approach, long acting GLP-1 compoundssuitable for the treatment regimen of the present invention can bederivitized. Derivatization is accomplished by various means.Modifications at amino acid side groups include, without limitation,acylation of lysine s-amino groups, N-alkylation of arginine, histidine,or lysine, alkylation of glutamic or aspartic carboxylic acid groups,and deamidation of glutamine or asparagine. Modifications of theterminal amino group include, without limitation, the des-amino, N-loweralkyl, N-di-lower alkyl, and N-acyl modifications. Modifications of theterminal carboxy group include, without limitation, the amide, loweralkyl amide, dialkyl amide, and lower alkyl ester modifications.Furthermore, one or more side groups, or terminal groups, may beprotected by protective groups known to the ordinarily-skilled proteinchemist. The α-carbon of an amino acid may be mono- or dimethylated.

[0129] Preferred GLP-1 derivatives are achieved through acylation. Usingthe principle of fatty acid derivitization, GLP-1 action is protractedby facilitating binding to plasma albumin via association of the fattyacid residue to fatty acid binding sites on albumin in the blood andperipheral tissues. A preferred GLP-1 derivative isArg³⁴Lys²⁶-(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37). GLP-1derivatives and methods of making such derivatives are disclosed inKnudsen, et al. (2000) J. Med. Chem. 43:1664-1669. In addition, numerouspublished applications describe derivatives of GLP-1, GLP-1 analogs,Exendin-4, and Exendin-4 analogs. See U.S. Pat. No. 5,512,540,WO96/29342, WO98/08871, WO99/43341, WO99/43708, WO99/43707, WO99/43706,and WO99/43705.

[0130] GLP-1 peptides can also be encapsulated using microspheres andthen delivered orally. For example, GLP-1 compounds can be encapsulatedinto microspheres composed of a commercially available, biocompatible,biodegradable polymer, poly(lactide-co-glycolide)-COOH and olive oil asa filler. See Joseph, et al. (2000) Diabetologia 43:1319-1328. Othertypes of microsphere technology is also available commercially such asMedisorb® and Prolease® biodegradable polymers from Alkermes. Medisorb®polymers can be produced with any of the lactide isomers.Lactide:glycolide ratios can be varied between 0:100 and 100:0 allowingfor a broad range of polymer properties. This allows for the design ofdelivery systems and implantable devices with resorption times rangingfrom weeks to months.

[0131] Another embodiment of the present invention encompasses articlesof manufacture for human pharmaceutical use comprising a package insert,a container, and said insert describing a treatment regimen whichinvolves maintaining plasma levels of a GLP-1 compound with a particularpotency within a certain range that avoids or minimizes side effectssuch as nausea and vomiting.

[0132] The container used in the present article of manufacture isconventional in the pharmaceutical arts. Generally, the container is avial or cartridge, usually made of glass, and accompanying cap, closure,bead, plunger, septum, and/or seal or other such article suitable foruse by the patient or pharmacist. Alternatively, the container is partof a kit consisting of a cartridge containing dried powder and a syringepre-filled with an appropriate diluent. Other options include thecontainer consisting of a dual chamber cartridge with a bypass thatkeeps the diluent liquid and the dried powder separate from each otheruntil reconstitution is desired. At the time of reconstitution, the dualchamber cartridge permits the diluent liquid to flow into the driedpowder. Preferably, the container is sized to accommodate 0.1 to 100 mL,preferably 0.5 to 25 mL, and more preferably, 5 to 10 mL, even morepreferably 1.5 to 3 mL volumes. Alternatively, the container is ablister, capsule, or blister disc. Other options for the containerinclude a transdermal patch, implantable device, microsphere carriersand other depot delivery systems.

[0133] The insert may provide the physician with a choice of severaldoses which result in plasma levels of the GLP-1 compound within theranges described herein, or preferably the insert will provide thephysician with a single dose which results in plasma levels of the GLP-1compound within the ranges described herein.

[0134] The package insert provides a description of how to administer apharmaceutical product, along with the safety and efficacy data requiredto allow the physician, pharmacist, and patient to make an informeddecision regarding the use of the product. The package insert generallyis regarded as the label of the pharmaceutical product.

[0135] The package insert may provide some or all of the followingindications or label descriptions:

[0136] 1) improved glycemic control in patients inadequately controlledon single or multiple oral anti-diabetic agents as a monotherapy or as acombination therapy with single of multiple oral anti-diabetic agentscompared to such agents alone;

[0137] 2) use for patients with inadequately controlled hyperglycemia;

[0138] 3) mean reduction in HbAlc greater than or equal to 0.5%,preferably greater than or equal to 1% in patients inadequatelycontrolled on single or multiple oral anti-diabetic agents;

[0139] 4) mean weight gain for patients on monotherapy will be less thanthe mean weight gain for patients treated with either a TZD orsulfonylurea as monotherapy over a 3 month period or a longer period;

[0140] 5) statistically significant demonstration of weight loss;

[0141] 6) no severe hypoglycemia at therapeutic dose;

[0142] 7) no symptomatic hypoglycemia at therapeutic dose;

[0143] 8) no fixed injection meal interval;

[0144] 9) initiation of daily dosing requires no more than moderate dosetitration (less than or equal to 4 doses) with subsequent daily dosingindependent of blood glucose monitoring;

[0145] 10) at least 12 months, preferably at least 18 monthsrefrigerated shelf-life;

[0146] 11) room temperature in-use storage;

[0147] 12) minimal injection site discomfort at therapeutic dose;

[0148] 13) no injection site discomfort at therapeutic dose;

[0149] 14) minimal nausea at therapeutic dose;

[0150] 15) β cell preservation in animal models;

[0151] 16) β cell preservation in humans;

[0152] 17) injection volume between 0.1 and 0.25 mLs; and

[0153] 18) safe for use in children.

[0154] Furthermore, the package insert may provide instructionsregarding the treatment regimen encompassed by the present inventioninvolving maintaining continuous plasma levels of GLP-1 within atherapeutic range regardless of the patient's body weight or body massindex, sex, or age. In addition, the package insert describes how thepresent invention provides a means to maintain steady state GLP-1 levelswith a protocol that does not require the patient to self-monitorglucose levels, and that does not need to be timed with meals therebyallowing patient convenience while safely maintaining optimal bloodglucose control.

[0155] Incidences of side effects are notably reduced due to thepresently claimed article of manufacture providing a chronic dosingregimen. Therefore, the preferred article of manufacture provides apackage insert having reported incidences of nausea in less than 30% ofpatients with plasma levels within the ranges described herein. Morepreferably, nausea and vomiting occurs in less than 20% of patients withplasma levels within the ranges described herein. Even more preferably,less than 10% of patients with plasma levels within the ranges describedherein experience such side effects. Most preferably, nausea andvomiting occur in less than 5% of patients with plasma levels within theranges described herein.

[0156] Incidences of hypoglycemia due to the treatment regimen describedherein are rare. The package insert having reported incidences ofhypoglycemia characterized by a blood glucose level less than 63 mg/dLis less than 10%, preferably less than 5%, and most preferably there areno reports of hypoglycemia.

[0157] The invention is illustrated by the following examples which arenot intended to be limiting in any way.

EXAMPLE 1

[0158] Clinical Study in Type 2 Diabetics

[0159] Four groups of eight type 2 diabetic patients were treated with along-acting formulation of Val⁸-GLP-1(7-37)OH. The first three groupsreceived either 2.5 or 3.5 or 4.5 mg once a day for 6 days. The fourthgroup received 4.5 mg once per day for 21 days. On the day before thestudy, each patient received a saline injection as placebo. Bloodglucose was followed for 13 hours. All meals during the evaluation dayswere strictly standardized. Patients were outpatients except for the Day6 and Day 21 evaluations over 24 hours. Following the injection on Day1, blood samples were taken for glucose and Val⁸-GLP-1(7-37)OH plasmalevels during 4 hours. Patients were dosed each morning. On the sixthday of dosing (and also Day 21 for Group 4), samples were collected upto 26 hours post dose for the blood glucose and Val⁸-GLP-1(7-37)OHplasma level determinations. Val⁸-GLP-1(7-37)OH plasma levels arerepresented in FIGS. 1 and 3 and corresponding glucose levels arerepresented in FIGS. 2 and 4. Patients in the 21 day dosing group lostan average of 2.125 kg (standard deviation: 1.36 kg). Five subjects losta total of 3 kg, one lost 2 kg, and 2 lost no weight.

EXAMPLE 2

[0160] Determination of Val⁸-GLP-1(7-37)OH Plasma Levels:

[0161] Due to the presence of endogenous concentrations of native GLP-1peptides and degradation products such as GLP-1 (9-37)OH by DPP-IV,concentrations of intact Val⁸-GLP-1(7-37)OH were measured using an ELISAassay in which full-length non-degraded Val⁸-GLP-1(7-37)OH isspecifically recognized. Immunoreactive Val⁸-GLP-1(7-37)OH is capturedfrom the plasma by an N-terminal anti-Val⁸-GLP-1(7-37)OH specificantisera immobilized onto a microtiter plate. This antisera is highlyspecific to the N-terminus of Val⁸-GLP-1(7-37)OH. Analkaline-phosphatase conjugated antibody, specific for the C-terminus ofGLP-1, is added to complete the “sandwich.” Detection is completed usingpNPP, a colormetric substrate for alkaline phosphatase. The amount ofcolor generated is directly proportional to the concentration ofimmunoreactive Val⁸-GLP-1(7-37)OH present in the sample. Quantitation ofVal⁸-GLP-1(7-37)OH in human plasma can be interpolated from a standardcurve using Val⁸-GLP-1(7-37)OH as the reference standard. Data wasanalyzed by a computer program using a weighted 4-parameter logisticalgorithm. The concentration of immunoreactive Val⁸-GLP-1(7-37)OH intest samples was determined using a standard curve.

EXAMPLE 3

[0162] In Vitro Potency Assay:

[0163] HEK-293 Aurora CRE-BLAM cells expressing the human GLP-1 receptorare seeded at 20,000 to 40,000 cells/well/100 μl into a 96 well blackclear bottom plate. The day after seeding, the medium is replaced withplasma free medium. On the third day after seeding, 20 μl of plasma freemedium containing different concentrations of GLP-1 agonist is added toeach well to generate a dose response curve. Generally, fourteendilutions containing from 3 nanomolar to 30 nanomolar GLP-1 compoundwere used to generate a dose response curve from which EC50 values couldbe determined. After 5 hours of incubation with GLP-1 compound, 20 μl ofβ-lactamase substrate (CCF2-AM—Aurora Biosciences—product code 100012)was added and incubation continued for 1 hour at which point theflourescence was determined on a cytoflour.

EXAMPLE 4

[0164] Crystallization of GLP-1(7-37)OH:

[0165] GLP-1(7-37)OH was dissolved in about 0.5 mL of 0.015 N NaOH at aconcentration of about 17 mg/mL, based on the mass of the peptide. Theprotein solution was adjusted to about pH 10.5 with dilute NaOH. Thesolution was held at ambient temperature for about 1 hour.

[0166] To a 390 μL aliquot of this peptide solution was added 25 μL of a1.0 M glycine pH 10 solution, giving a final concentration of about 16mg/mL of GLP-1(7-37)OH and about 60 mM glycine. The pH of the solutionwas adjusted to about pH 10 with dilute HCl and/or dilute NaOH asneeded.

[0167] The solution was then filtered into a glass vial through asterile 0.22 μm Millex®-GV (Millipore Corporation, Waltham, Mass., USA)4 mm filter unit that had been pre-rinsed with 60 mM glycine buffer atpH 10.

[0168] To 300 μL of the filtered peptide solution was added 66 μL of a50% ethanol solution in water. To this solution was added, in smallincrements, a total of 14.1 μL of a 150 mM zinc oxide pH 2.3 solution(prepared with dilute HCl), with mixing by hand performed after eachincrement was added until the solution became clear. The molar ratio ofzinc:peptide was about 1.5:1.

[0169] The final solution was adjusted to about pH 9.0 andcrystallization proceeded at ambient temperature. The crystallizationsolution comprised about 12.6 mg/mL GLP-1(7-37)OH, 47 mM glycine, 8.7%ethanol by volume, and about 1.5 moles of zinc per mole of GLP-1(7-37)OHat pH 9.0.

[0170] After 1 day at ambient temperature, thin plate crystals ofGLP-1(7-37)OH were observed under a microscope at 400× magnification.

[0171] The yield of crystallization was determined by using aspectrophotometer to compare the absorbance of an aliquot of the entiresuspension redissolved in a 10-fold dilution of 0.01N HCl, with asimilarly diluted supernatant obtained by centrifuging the suspensionfor about 4 minutes at 14,000×g. For this experiment, thecrystallization yield was 92%.

EXAMPLE 5

[0172] Crystallization of Val⁸-GLP-1(7-37)OH:

[0173] Val⁸-GLP-1(7-37)OH was dissolved in about 0.57 mL of 0.015 N NaOHat a concentration of about 17 mg/mL, based on the mass of the peptide.The protein solution was adjusted to about pH 10.5 with dilute NaOH. Thesolution was held at ambient temperature for about 1 hour.

[0174] To a 390 μL aliquot of this peptide solution was added 25 μL of a1.0 M glycine pH 8 solution, giving a final concentration of about 16mg/mL of Val⁸-GLP-1(7-37)OH and about 60 mM glycine. The pH of thesolution (about pH 9.0) was adjusted to about pH 9.9 with dilute HCland/or dilute NaOH as needed.

[0175] The solution was then filtered into a 0.5 mL Eppendorf tubethrough a sterile 0.22 μm Millex®-GV (Millipore Corporation, Waltham,Mass., USA) 4 mm filter unit.

[0176] To 300 μL of the filtered peptide solution in a clean test tubewas added 66 μL of a 50% ethanol solution in water. To this solution wasadded, in small increments, a total of 14.1 μL of a 150 mM zinc oxide pH2.3 solution (prepared with dilute HCl), with mixing by hand performedafter each increment was added until the solution became clear. Themolar ratio of zinc:peptide was about 1.5:1.

[0177] The final solution was adjusted to about pH 8.9 andcrystallization proceeded at ambient temperature. The crystallizationsolution comprised about 12.6 mg/mL Val⁸-GLP-1(7-37)OH, 47 mM glycine,8.7% ethanol by volume, and about 1.5 moles of zinc per mole ofVal⁸-GLP-1(7-37)OH at pH 8.9.

[0178] After about three days at ambient temperature, thin platecrystals of Val⁸-GLP-1(7-37)OH were observed under a microscope at 400×magnification.

EXAMPLE 6

[0179] Crystallization of Val⁸-GLP-1(7-36)NH₂:

[0180] Val⁸-GLP-1(7-36)NH₂ was dissolved in about 0.44 mL of 0.015 NNaOH at a concentration of about 17 mg/mL, based on the mass of thepeptide. The protein solution was adjusted to about pH 10.5 with diluteNaOH. The solution was held at ambient temperature for about 1 hour.

[0181] To a 390 μL aliquot of this peptide solution was added 25 μL of a1.0 M glycine pH 10.2 solution, giving a final concentration of about 16mg/mL of Val⁸-GLP-1(7-36)NH₂ and about 60 mM glycine.

[0182] The solution was then filtered into a glass vial through asterile 0.22 μm Millex®-GV (Millipore Corporation, Waltham, Mass., USA)4 mm filter unit.

[0183] To 300 μL of the filtered peptide solution in a clean glass vialwas added 66 μL of a 50% ethanol solution in water. To this solution wasadded, in small increments, a total of 14.1 μL of a 150 mM zinc oxide pH2.3 solution (prepared with dilute HCl), with mixing by hand performedafter each increment was added until the solution became clear. Themolar ratio of zinc:peptide was about 1.5:1.

[0184] The final solution was adjusted to about pH 9.85 andcrystallization proceeded at ambient temperature. The crystallizationsolution comprised about 12.6 mg/mL Val⁸-GLP-1(7-36)NH₂, 47 mM glycine,8.7% ethanol by volume, and about 1.5 moles of zinc per mole ofVal⁸-GLP-1(7-36)NH₂ at pH 9.85.

[0185] After about three days at ambient temperature, microcrystals ofVal⁸-GLP-1(7-36)NH₂ were observed under a microscope at 400×magnification.

EXAMPLE 7

[0186] Crystallization of Val⁸-GLP-1(7-37)NH₂:

[0187] Val⁸-GLP-1(7-37)NH₂ was dissolved in about 0.48 mL of 0.015 NNaOH at a concentration of about 17 mg/mL, based on the mass of thepeptide. The protein solution was adjusted to about pH 11.1 with diluteNaOH, then to pH 10.36 with dilute HCl. The solution was held at ambienttemperature for about 1 hour.

[0188] To a 390 μL aliquot of this peptide solution was added 25 μL of a1.0 M glycine pH 10 solution, giving a final concentration of about 16mg/mL of Val⁸-GLP-1(7-37)NH₂ and about 60 mM glycine.

[0189] The solution was then filtered into a glass vial through asterile 0.22 μm Millex®-GV (Millipore Corporation, Waltham, Mass., USA)4 mm filter unit.

[0190] To 300 μL of the filtered peptide solution in a clean glass vialwas added 66 μL of a 50% ethanol solution in water. To this solution wasadded, in small increments, a total of about 7 μL of a 150 mM zinc oxidepH 2.3 solution (prepared with dilute HCl), with mixing by handperformed after each increment was added until the solution becameclear. The molar ratio of zinc:peptide was about 0.75:1.

[0191] The final solution was adjusted to about pH 9.8 andcrystallization proceeded at ambient temperature. The crystallizationsolution comprised about 12.6 mg/mL Val⁸-GLP-1(7-37)NH₂, 47 mM glycine,8.7% ethanol by volume, and about 0.75 moles of zinc per mole ofVal⁸-GLP-1(7-37)NH₂ at pH 9.8.

[0192] After about 48 hours at ambient temperature, clusters ofVal⁸-GLP-1(7-37)NH₂ were observed under a microscope at 400×magnification.

1 7 1 31 PRT Homo sapiens MISC_FEATURE (31)..(31) Xaa at position 31 isGly or is absent. 1 His Gly Glu Gly Thr Phe Thr Ser Asp Val Ser Ser TyrLeu Glu Gly 1 5 10 15 Gln Ala Ala Lys Glu Phe Ile Ala Trp Leu Val LysGly Arg Xaa 20 25 30 2 39 PRT Artificial Sequence synthetic construct 2His Xaa Xaa Gly Thr Phe Thr Ser Asp Leu Ser Lys Gln Met Glu Glu 1 5 1015 Glu Ala Val Arg Leu Phe Ile Glu Trp Leu Lys Asn Gly Gly Pro Ser 20 2530 Ser Gly Ala Pro Pro Pro Ser 35 3 39 PRT Artificial Sequence syntheticconstruct 3 Xaa Xaa Xaa Gly Xaa Xaa Thr Xaa Asp Xaa Xaa Xaa Xaa Xaa XaaXaa 1 5 10 15 Xaa Xaa Xaa Xaa Xaa Phe Ile Xaa Xaa Xaa Xaa Xaa Xaa XaaXaa Xaa 20 25 30 Xaa Xaa Xaa Xaa Xaa Xaa Xaa 35 4 31 PRT ArtificialSequence synthetic construct 4 His Xaa Glu Gly Xaa Xaa Thr Ser Asp XaaSer Ser Tyr Leu Glu Xaa 1 5 10 15 Xaa Xaa Ala Xaa Xaa Phe Ile Xaa TrpLeu Xaa Xaa Xaa Xaa Xaa 20 25 30 5 31 PRT Artificial Sequence syntheticconstruct 5 His Xaa Glu Gly Thr Xaa Thr Ser Asp Xaa Ser Ser Tyr Leu GluXaa 1 5 10 15 Xaa Ala Ala Xaa Glu Phe Ile Xaa Trp Leu Val Lys Xaa ArgXaa 20 25 30 6 31 PRT Artificial Sequence synthetic construct 6 His XaaGlu Gly Thr Phe Thr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 1 5 10 15 XaaAla Ala Lys Xaa Phe Ile Xaa Trp Leu Val Lys Gly Arg Xaa 20 25 30 7 31PRT Artificial Sequence synthetic construct 7 Xaa Xaa Glu Gly Thr PheThr Ser Asp Val Ser Ser Tyr Leu Glu Xaa 1 5 10 15 Gln Ala Ala Lys GluPhe Ile Ala Trp Leu Val Lys Gly Arg Xaa 20 25 30

We claim:
 1. A method of normalizing blood glucose levels whichcomprises maintaining chronic steady state plasma levels between about60 picomoles/liter and about 200 picomoles/liter of a GLP-1 analog orderivative in biologically active form having an in vitro potency withintwo-fold the in vitro potency of Val⁸-GLP-1(7-37)OH wherein the GLP-1analog or derivative is administered by subcutaneous injection to ahuman subject no more than once or twice every 24 hours.
 2. A method oftreating a condition selected from the group consisting of:hyperglycemia, type 2 diabetes, obesity, stroke, myocardial infarction,catabolic changes that occur after surgery, and irritable bowel syndromewhich comprises maintaining chronic steady state plasma levels betweenabout 60 picomoles/liter and about 200 picomoles/liter of a GLP-1 analogor derivative in biologically active form having an in vitro potencywithin two-fold the in vitro potency of Val⁸-GLP-1(7-37)OH wherein theGLP-1 analog or derivative is administered by subcutaneous injection toa human subject no more than once or twice every 24 hours.
 3. A methodof preventing β-cell deterioration which comprises maintaining steadystate chronic plasma levels between about 60 picomoles/liter and about200 picomoles/liter of a GLP-1 analog or derivative in biologicallyactive form having an in vitro potency within two-fold the in vitropotency of Val⁸-GLP-1(7-37)OH wherein the GLP-1 compound is administeredby subcutaneous injection to a human subject no more than once or twiceevery 24 hours.
 4. A method of inducing weight loss which comprisesmaintaining chronic steady state plasma levels between about 60picomoles/liter and about 200 picomoles/liter of a GLP-1 analog orderivative in biologically active form having an in vitro potency withintwo-fold the in vitro potency of Val⁸-GLP-1(7-37)OH wherein the GLP-1compound is administered by subcutaneous injection to a human subject nomore than once or twice every 24 hours.
 5. The method of any one ofclaims 1 through 4 wherein the plasma levels are maintained betweenabout 100 picomolar and about 200 picomolar.
 6. The method of claim 5wherein the plasma levels are maintained between about 100 picomolar andabout 180 picomolar.
 7. A method of normalizing blood glucose levelswhich comprises maintaining chronic steady state plasma levels betweenabout 60/X picomolar and about 200/X picomolar of a GLP-1 analog orderivative in biologically active form wherein X is the in vitro potencyof the GLP-1 analog or derivative relative to Val⁸-GLP-1(7-37)OH whichis given a reference value of 1 and wherein the GLP-1 analog orderivative is administered by subcutaneous injection to a human subjectno more than once or twice every 24 hours.
 8. A method of treating acondition selected from the group consisting of: hyperglycemia, type 2diabetes, obesity, stroke, myocardial infarction, catabolic changes thatoccur after surgery, and irritable bowel syndrome which comprisesmaintaining chronic steady state plasma levels between about 60/Xpicomolar and about 200/X picomolar of a GLP-1 analog or derivative inbiologically active form wherein X is the in vitro potency of the GLP-1analog or derivative relative to Val⁸-GLP-1(7-37)OH which is given areference value of 1 and wherein the GLP-1 analog or derivative isadministered by subcutaneous injection to a human subject no more thanonce or twice every 24 hours.
 9. A method of preventing β celldeterioration which comprises maintaining chronic steady state plasmalevels between about 60/X picomolar and about 200/X picomolar of a GLP-1analog or derivative in biologically active form wherein X is the invitro potency of the GLP-1 analog or derivative relative to Val⁸-GLP-1(7-37)OH which is given a reference value of 1 and wherein the GLP-1analog or derivative is administered by subcutaneous injection to ahuman subject no more than once or twice every 24 hours.
 10. A method ofinducing weight loss which comprises maintaining chronic steady stateplasma levels between about 60/X picomolar and about 200/X picomolar ofa GLP-1 analog or derivative in biologically active form wherein X isthe in vitro potency of the GLP-1 analog or derivative relative toVal⁸-GLP-1(7-37)OH which is given a reference value of 1 and wherein theGLP-1 analog or derivative is administered by subcutaneous injection toa human subject no more than once or twice every 24 hours.
 11. Themethod of any one of claims 7 through 10 wherein the plasma levels aremaintained between about 100/X picomolar and about 200/X picomolar. 12.The method of claim 11 wherein the plasma levels are maintained betweenabout 100/X picomolar and about 180/X picomolar.
 13. The method of anyone of claims 1 through 12 wherein the GLP-1 analog or derivative isadministered not more than once every 24 hours.
 14. The method of anyone of claims 1 through 6 wherein the GLP-1 analog or derivative isselected from the group consisting of: Val⁸-GLP-1(7-37)OH,Val⁸-GLP-1(7-36)NH₂, Gly⁸-GLP-1(7-37)OH, Gly⁸-GLP-1(7-36)NH₂,Val⁸-Lys²²-GLP-1(7-37)OH Val⁸-Lys²²-GLP-1(7-36)NH₂,Val⁸-Glu³⁰-GLP-1(7-37)OH, Val⁸-Glu³⁰-GLP-1(7-36)NH₂,Gly⁸-Glu³⁰-GLP-1(7-37)OH, Gly⁸-Glu³⁰-GLP-1(7-36)NH₂,Val⁸-His³⁷-GLP-1(7-37)OH, and Val⁸-His³⁷-GLP-1(7-36)NH₂,Arg³⁴-GLP-1(7-36)NH₂, Arg³⁴-GLP-1(7-37)OH.
 15. The method of any one ofclaims 1 through 6 wherein the GLP-1 analog or derivative is a GLP-1analog which is administered as a crystal suspension formulation. 16.The method of any one of claims 1 through 14 wherein the GLP-1 analog orderivative is an acylated GLP-1 derivative.
 17. The method of claim 16wherein the acylated GLP-1 derivative is a GLP-1 analog acylated at theepsilon-amino group of lysine present at position
 26. 18. The method ofclaim 17 wherein the acylated GLP-1 derivative isArg³⁴Lys²⁶-(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37).
 19. A method ofnormalizing blood glucose levels which comprises maintaining chronicsteady state plasma levels between about 6 picomoles/liter and about 40picomoles/liter of Exendin-4 wherein the Exendin-4 is administered bysubcutaneous injection to a human subject.
 20. A method of treating acondition selected from the group consisting of: hyperglycemia, type 2diabetes, obesity, stroke, myocardial infarction, catabolic changes thatoccur after surgery, and irritable bowel syndrome which comprisesmaintaining chronic steady state plasma levels between about 6picomoles/liter and about 40 picomoles/liter of Exendin-4 wherein theExendin-4 is administered by subcutaneous injection to a human subject.21. A method of preventing β-cell deterioration which comprisesmaintaining steady state chronic plasma levels between about 6picomoles/liter and about 40 picomoles/liter of Exendin-4 wherein theExendin-4 is administered by subcutaneous injection to a human subject.22. A method of inducing weight loss which comprises maintaining chronicsteady state plasma levels between about 6 picomoles/liter and about 40picomoles/liter of Exendin-4 wherein the Exendin-4 is administered bysubcutaneous injection to a human subject.
 23. The method of any one ofclaims 1 through 38 wherein the GLP-1 reaches steady state plasma levelsafter six days of chronic once a day dosing.
 24. The method of claim 23wherein the GLP-1 compound accumulates approximately three-fold in theplasma after six days of chronic once a day dosing.
 25. An article ofmanufacture for human pharmaceutical use comprising: a) a container; b)a dosage form comprising an amount of a GLP-1 analog or derivativehaving an in vitro potency within two-fold that of Val⁸-GLP-1(7-37)OH;and c) a package insert that provides for administration of the dosageform that results in maintaining GLP-1 compound plasma levels between 60picomoles and 200 picomoles.
 26. The article of claim 25 wherein thepackage insert directs use to treat a condition selected from the groupconsisting of: hyperglycemia, type 2 diabetes, stroke, myocardialinfarction, catabolic changes that occur after surgery, obesity, andirritable bowel syndrome.
 27. The article of claim 25 or 26 wherein theGLP-1 analog or derivative is selected from the group consisting ofVal⁸-GLP-1(7-37)OH andArg³⁴Lys²⁶-(N-ε-(γ-Glu(N-α-hexadecanoyl)))-GLP-1(7-37).
 28. An articleof manufacture for human pharmaceutical use comprising: a) a container;b) a dosage form comprising an amount of Exendin-4; and c) a packageinsert that provides for administration of the dosage form that resultsin maintaining Exendin-4 plasma levels between 6 picomoles and 40picomoles.
 29. Use of a GLP-1 analog or derivative having an in vitropotency within 2-fold that of Val⁸-GLP-1(7-37)OH for the manufacture ofa medicament for normalizing blood glucose, preserving β cells, inducingweight loss, or treating a condition selected from the group consistingof: hyperglycemia, type 2 diabetes, stroke, myocardial infarction,catabolic changes that occur after surgery, obesity, and irritable bowelsyndrome which comprises maintaining chronic steady state plasma levelsof the GLP-1 analog or derivative between about 60 picomolar and about200 picomolar and wherein the GLP-1 analog or derivative is administeredby subcutaneous injection to a human subject not more the once or triceevery 24 hours.
 30. The Use of claim 29 wherein the plasma levels aremaintained between about 100 picomolar and about 200 picomolar.
 31. Useof Exendin-4 for the manufacture of a medicament for normalizing bloodglucose, preserving β cells, inducing weight loss, or treating acondition selected from the group consisting of: hyperglycemia, type 2diabetes, stroke, myocardial infarction, catabolic changes that occurafter surgery, obesity, and irritable bowel syndrome which comprisesmaintaining chronic steady state plasma levels of Exendin-4 betweenabout 6 picomolar and about 40 picomolar and wherein the Exendin-4 isadministered by subcutaneous injection to a human subject.
 32. Use of aGLP-1 analog or derivative for the manufacture of a medicament fornormalizing blood glucose, preserving β cells, inducing weight loss, ortreating a condition selected from the group consisting of:hyperglycemia, type 2 diabetes, stroke, myocardial infarction, catabolicchanges that occur after surgery, obesity, and irritable bowel syndromewhich comprises maintaining chronic steady state plasma levels of theGLP-1 analog or derivative between about 60/X picomolar and about 200/Xpicomolar wherein X is the in vitro potency of the GLP-1 analog orderivative relative to Val⁸-GLP-1(7-37)OH which is given a referencevalue of 1 and wherein the GLP-1 analog or derivative is administered bysubcutaneous injection no more than once or twice every 24 hours.